- Email: [email protected]
Control of globin synthesis by hemin: Factors influencing formation of an inhibitor of globin chain initiation in reticulocyte lysates
34 °
BIOCHIMICA ET BIOPHYSICA ACTA
8BA 97465
CONTROL OF GLOBIN S Y N T H E S I S BY H E M I N : FACTORS I N F L U E N C I N G FORMATION OF AN I N H I B I T O R OF G L O B I N CHAIN I N I T I A T I O N IN R E T I C U L O C Y T E LYSATES M A R T I N G R O S S " AND M A R C O R A B I N O V I T Z
Laboratory of Physiology, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.) ( R e c e i v e d J u n e i 2 t h , 1972)
S U MMARY
The control of globin synthesis by hemin in rabbit reticulocyte lysates is mediated by the formation of an inhibitor of globin chain initiation. This cytoplasmic repressor does not appear to be a product derived from newly synthesized globin. It is formed in the absence of hemin from a protein in the post-ribosomal fraction without participation of free, low molecular weight components. This proinhibitor and the inhibitor are eluted at the same point on a Sephadex G-2oo column, which corresponds to a molecular weight of 3.0 • lO 5. Although the complete transition of proinhibitor to inhibitor in the ribosome-free supernatant fraction takes place over a period of 12 to 15 h, partially purified proinhibitor can be converted to inhibitor within 15 min b y reaction with N-ethylmaleimide or o-iodosobenzoate. These findings suggest that inhibitor formation m a y involve a specific conformational change in the proinhibitor molecule.
INTRODUCTION
The maintenance of globin synthesis in the reticulocyte is dependent upon the availability of hemin 1'2. In the absence of iron for heroin synthesis 3 or of heroin itself 3-5, the cellular polyribosomes disaggregate, indicating that control is at the site of chain initiation e. Reticulocyte lysates are similarly dependent upon heroin for sustained globin synthesis 7-9. This regulation is mediated through the suppression by heroin of formation of an inhibitor of globin chain initiation in the post-ribosomal supernatant fraction 1°-15. Within 30 rain of its formation, this inhibitor, a translational repressor, can be completely inactivated by heroin and is denoted as reversible inhibitor ~5. With longer incubations in the absence of heroin, the inhibitory activity can no longer be overcome by hemin and the material formed is denoted as irreversible inhibitor 10-15. In this report we describe experiments designed to study the nature of the precursor of the inhibitor, termed proinhibitor, the protective effect of heroin, and the reaction involving transformation of proinhibitor to inhibitor. The formation of irreversible inhibitor was followed since it can be quantitatively assayed and appears to be the ultimate product of the reactions being studied. A b b r e v i a t i o n : H E P E S , N-e-hydroxyethylpiperazine-N'-2-ethanesulfoniatc. * P r e s e n t a d d r e s s : D e p a r t m e n t of P a t h o l o g y , U n i v e r s i t y of Chicago, Chicago, Ill., U.S.A.
Biochim. Biophys. Acta, 287 (i972) 34o-352
INHIBITOR OF GLOBIN CHAIN INITIATION
34 [
METHODS
Sources o/materials The sources of materials required for the cell-free system were indicated previously n,13. N-Ethylmaleimide and o-iodosobenzoate were purchased from Sigma, and dithiothreitol, from CalBiochem. H E P E S (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid) and horse spleen apoferritin were from CalBiochem, and pyruvate kinase, from Worthington. Sephadex G-25, G-5o, and G-2oo, blue dextran 2000, and CM-Sephadex C-5o were from Pharmacia, and Bio-Gel P-I5O, from Bio-Rad. Aquasol was purchased from New England Nuclear.
Cell-/ree incubations and assay/or inhibitor The method of preparation of reticulocyte lysate from phenylhydrazine-treated rabbits has been described 18. Incubation mixtures contained 5 vol. of lysate, 3 vol. of master mix, and 3 vol. of water or of a solution of the material being studied. Unless otherwise indicated, heroin was added so that the final concentration was 20/*M. The assay of the inhibitor was carried out as previously describedlS; one unit of inhibitor being defined as that amount which reduces to 5 ° % the stimulation of protein synthesis by hemin in a standard incubation system. Master mix contained the following components in amounts to yield the indicated final concentration in the total reaction mixture: KC1 (75 mM); MgC1, (2 nlM); ATP (0.5 mM); GTP (0.2 raM); creatine phosphate (15 raM); creatine phosphokinase (45 units/ml); L-[14C]leucine (0.3 mM, 5/*Ci/#mole) and the other 19 amino acids at concentrations corresponding to the amino acid composition of rabbit hemoglobin 16. Unless otherwise indicated, incubations were carried out at 34 °C for 9 ° rain, and the hot trichloroacetic acidinsoluble radioactivity in 25-/.1 aliquots of the total reaction mixture was determined by plating on Millipore filters and counting in a gas-flow counter with Micromil window. The post-ribosomal supernatant fraction (denoted supernate) was prepared by centrifuging lysate in cut-down tubes at approximately 400 ooo × g for 60 min in the SW-56 rotor 17.
Chromatography o/the products o/the cell-]ree reaction on Biogel P-z5o All preparative and chromatographic procedures were performed at o to 4 °C unless otherwise indicated. The post-ribosomal fraction, (3-3 ml), prepared from the total reaction mixture by centrifugation at 3oo ooo × g (average) for 2 h in the SW56 rotor, was applied to the top of a 145 cm × 2.5 cm Biogel P-I5O column equilibrated with 3.7 mM H E P E S (pH 7.2) and o.o275 M KC1 (Buffer A) and eluted under gravity flow with this buffer. The flow rate was 15 ml/h, and 3.5-ml fractions were collected. The radioactivity in fractions not containing hemoglobin was determined on o.5o-ml aliquots in IO ml Aquasol with a Packard model No. 4322 liquid scintillation spectrometer (efficiency 9 ° %). Diluted aliquots of the fractions containing hemoglobin were evaporated to dryness on planchets and counted in a Nuclear Chicago gas-flow counter with Mieromil window (efficiency 34 %). Radioactivity determined by the latter method was corrected to 9 ° % efficiency to permit direct comparison with fractions counted on the scintillation spectrometer.
Preparation o/ proinhibitor on CM-Sephadex Large-scale preparation of proinhibitor was accomplished by applying 20 ml Biochim. Biophys. Acta, 287 (i972) 34o-352
342
M. GROSS, M. RABINOVITZ
of fresh supernate to a 4 ° cm × 4.5 cm CM-Sephadex colunm equilibrated with 3.7 mM H E P E S , pH 7.2 (Buffer B). Elution was with Buffer B, and the region capable of forming inhibitor, which corresponds to the void volume of the column, was pooled and lyophilized. The lyophilized product was dissolved in a volume of water equivalent to one-third or one-fifth the volume of the original supernate and stored at --8o °C. It is denoted as CMS-proinhibitor.
Determination o/molecular weight o/ proinhibitor and o/inhibitor on a Sephadex G-2oo column A 14o cm × 2.5 cm Sephadex G-2oo column, equilibrated with Buffer A, was developed with this buffer at a rate of 16 ml/h. The indicated markers were each applied separately to the column in 4.0 ml, fractions containing 4.0 ml were collected, and the elution volume was determined by the peak of absorbance at 280 nm (apoferritin and pyruvate kinase), 260 nm (blue dextran), or 415 nm (rabbit hemoglobin). The location of inhibitor was determined as the peak of activity after the application of I ml of inhibitory supernate (3000 units) mixed with 3 ml of water. To locate proinhibitor, 4.0 ml of fresh supernate were applied to the column and the capacity to form inhibitor was determined by measuring inhibitory activity after warming each column fraction at 34 °C for 4 h.
Preparation o] inhibitory activity/rom CMS-proinhibitor with sul/hydryl reagents I vol. of CMS-proinhibitor, concentrated to one-third the volume of the original supernate, was mixed with i vol. of either IO mM N-ethylmaleimide or 25 mM o-iodosobenzoate, both prepared in IO times concentrated Buffer A. After incubation for 5 min at 34 °C, the N-ethylmaleimide in the treated sample was neutralized with I vol. of 5 mM dithiothreitol in IO times concentrated Buffer A. After 2 min incubation at 34 °C, the o-iodosobenzoate in the treated sample was neutralized with i vol. of 25 mM dithiothreitol. After incubation of both samples at 34 °C for a total of 15 min, they were cooled to o °C and used immediately. Inhibitor formed by incubation of CMS-proinhibitor with N-ethylmaleimide and o-iodosobenzoate is denoted as N-ethylmaleimide-inhibitor and o-iodosobenzoate-inhibitor, respectively.
RESULTS
The inhibitor is not derived/rom newly/ormed globin The inhibitor which appears when post-ribosomal supernate of a rabbit reticulocyte lysate is warmed without heroin, is a protein 14,1s which is derived from a preformed proinhibitor 1~. Since reticulocytes contain a pool of free :c-chains which are unstable in the absence of hemin 19-*t, we tested whether the proinhibitor was a form of globin or was derived from globin. Taking advantage of the fact that there is a long lag in the formation of inhibitor at 25 °C (ref. I3), proteins of a lysate were labeled with [~*Cjleucine in the absence of hemin at this temperature. The products of the reaction, freed of ribosomes, were analyzed on a Biogel P-I50 column either immediately (Fig. I) or after warming at 34 °C (Fig. 2). In the former, a small amount of radioactivity and inhibitory activity was eluted with the void volume. About onehalf of the remaining radioactivity was coincident with hemoglobin, and the other
Biochirn. Biophys. Acta, 287 (1972) 340-352
343
INHIBITOR OF GLOBIN CHAIN INITIATION ~
90
~a
.~
A
{6o 75
5
06
5
I
i\
cz:o
-
\
f~'
0.2~
(n
.... 77 50
60
70 FRACTION NUMBER
80
90
I00
I10
Fig. I. Gel filtration of s u p e r n a t e containing proinhibitor and labeled globin prior to the formation of inhibitor. An incubation mixture, containing 3.o ml of lysate w i t h o u t added hemin, 1.8 ml of m a s t e r mix and 1.8 ml of water, was incubated a t 25 °C for 60 min. The ribosomes were t h e n r e m o v e d b y centrifugation, the s u p e r n a t e fractionated on a Biogel P - i 5 o c o l u m n and the inhib i t o r y p o t e n c y a n d r a d i o a c t i v i t y of fractions were determined as described in Methods. The abscissa has been divided to p e r m i t a change in scale for b o t h radioactivity and absorbance.
0
?-
9O
&
25
! i
o
_o
i .
c
~ 5o
~o
it
60
il
75
o
0.6
g 2m~~ 2 §.~
O
Z i
02 i
~5
o
g J I
50
60
70
80
90
I00
IiO
FRACTION NUMBER
Fig. 2. Gel filtration of s u p e r n a t e containing p r o i n h i b i t o r and labeled globin after f o r m a t i o n of inhibitor. After the r e m o v a l of ribosomes, the s u p e r n a t e was incubated at 34 °C for 16 h before fractionation on a Biogel P-I5O column. Conditions are as indicated in the legend to Fig. I.
half was eluted at a position corresponding to a molecular weight of about 33 ooo. The latter component was mixed with unlabeled rabbit hemoglobin, hemin was split from globin with acid-acetone, and ~- and fl-chains were separated by carboxymethyl cellulose chromatography 2z,23. Under these conditions 95 % of the labeled protein co-chromatographed with the ~-chain. When the products of the cell-free reaction were separated on Biogel P-I5O after warming at 34 °C (Fig. 2), additional inhibitory Biochim. Biophys. Acta, 287 (1972) 340-352
344
M. GROSS, M. RABINOVITZ
activity was eluted with the void volume, but the amount of radioactivity in this region decreased as a result of the incubation. The labeled ~-globin fraction was found in a component which was eluted with hemoglobin and was not in the inhibitor region. The results suggest that the inhibitor is not derived from newly synthesized 0~-globin.
The molecular size o/the components o/ the inhibitory/orming system To determine whether any low molecular weight component is required for inhibitor formation, fresh, ribosome-free supernate was chromatographed on Sephadex G-25 and G-5o columns, and the fraction which was eluted at the void volume was warmed to determine its ability to form inhibitor. In the course of these studies, we found that the eluate from Sephadex G-25 columns was inhibitory before it was warmed. If, however, such a column was pretreated by an initial passage of the super-
8~
A
I
£-
i
200
40 4-
i
~5o
I|
I 12~
i
B
~c ~z
2C o
n~
N
9
~m Io 5=
o
1
m~
5O
2 I
2
5
4
5
6
7
HOURS
I 40
50
FRACTION NUMBER
Fig. 3. Inhibitor-forming capacity of the void volume eluate from supernates filtered on Scphadex G-25, Sephadex G-5 o, and Biogel P-I5O columns. (A) Fresh supernate (ioo/~1) was applied to i8 cm × 0.6 cm Sephadex G-25 ([], It) and Sephadex G-5o (&, A) columns, equilibrated with Buffer A. The Sephadex G-25 column h a d been pretreated with 2o0/~1 of fresh, normal rabbit hemolysate a n d t h e n washed t h o r o u g h l y with Buffer A. E a c h column was developed with Buffer A, and the void volume fractions, containing hemoglobin as a marker, were pooled. These eluates and an aliquot of the original s u p e r n a t e (O, O) were warmed at 34 °C for the indicated times in the presence (closed symbols) and absence (open symbols) of 35/2M hemin. Inhibitor activity was m e a s u r e d in duplicate samples, a n d the results are expressed as units/ml corrected to the concentration in the original supernate. (B) The pooled inhibitor-forming activity from the Biogel P-I5O column shown in Fig. 4 (Fractions 30-35) was lyophilized and t h e n reconstituted with water to the concentration of the original supernate. Aliquots of this P-I5O fraction (A, A) a n d of the original s u p e r n a t e (O, • ) were t h e n warmed at 34 °C in the presence (closed symbols) and absence (open symbols) of 35/~M hemin. Duplicate samples were removed at the indicated times for the determination of inhibitor activity. Fig. 4. C h r o m a t o g r a p h y of proinhibitor on Biogel P-i5o. Fresh supernate (6 ml) was applied to a i oo cm × 2.5 cm Biogel P-15o column equilibrated with Buffer A, and the column was developed with Buffer A a t a rate of i i ml/h; 3.5-ml fractions were collected. Inhibitory activity of the fractions was determined in duplicate; before incubation ( & . . . &), after incubation for 4 h at 34 °C ( O - - - O ) .
Biochim. Biophys. Mcta, 287 (1972) 34o-352
INHIBITOR OF GLOBIN CHAIN INITIATION
345
nate followed by thorough washing with buffer, the void volume eluate obtained when a second aliquot of supernate was fractionated was not inhibitory. This indicated that the Sephadex G-25 contained some material, inhibitory to the reticulocyte cell-free system, which could be removed by a concentrated hemolysate solution. Consequently, columns of Sephadex G-25 were routinely treated with 200 #1 of fresh, stroma-free hemolysate from non-anemic rabbits and then washed with the elution buffer. When fresh supernate was chromatographed on either Sephadex G-25 or G-5o (Fig. 3, Frame A), the void volume eluates formed inhibitor at the same rate as the original supernate. Hemin markedly retarded this reaction in all cases. When fresh supernate was fractionated on a Biogel P-i5o column (Fig. 4), inhibitor formation, determined by warming and then assaying individual fractions for inhibitory activity, was limited to the void volume region. The rate of inhibitor formation and the degree of suppression of the reaction by hemin is the same in this Biogel P-I5O preparation as it is in fresh supernate (Fig. 3, Frame B). The results indicate that no freely dissociable, low molecular weight component is required for inhibitor formation. The molecular weights of the proinhibitor and inhibitor were estimated with a calibrated Sephadex G-2oo column (Fig. 5). Both the proinhibitor and inhibitor were eluted similarly, at an elution volume corresponding to a molecular weight of approximately 3 " lO5. This suggests that there is no detectable size change in the transformation from proinhibitor to inhibitor. Adamson et al. ~4 have obtained a molecular weight of 5 " lO5 for the inhibitor by gel chromatography on Sepharose 6B. We have also obtained a higher molecular weight estimate using Sepharose 6B in place of Sephadex G-2oo.
2~
lit
o.5 2r~
I 300
t It
I I 3,~o 400 450 ELUTION VOLUME {roll
it,, I
~
~
Fig. 5. Determination of molecular weight of proinhibitor and of inhibitor on a Sephadex G-2oo column. Experimental conditions are given in Methods. Biochim. Biophys. Acta, 287 (1972) 340-352
346
i.
GROSS, M. RABINOV1TZ
The nature o/heroin protection The proinhibitor was prepared free of hemoglobin by fractionation of the supernate on a CM-Sephadex column. It was eluted in the void volume before the low molecular weight components, while hemoglobin was adsorbed to the top of the column and could be eluted with I M KC1 (Fig. 6). The proinhibitor was concentrated by lyophilization as described in Methods and denoted CMS-proinhibitor.
T b 20
IM
Il
iCt
,o /
Az6° = 0183 A28o " 12(3
--
I00 4= 80 o~
E~$0 = 1.05 -60
A~
iN 0i
A260 = 38 /Az8°
05
O
~
115 20 FRACTION NUMBER
-
",20 i ;o t o
-
I 20
I I I ~ 40 60 80 I00 HEMIN CONCENTRATION (FM)
_y~__
200
Fig. 6. C h r o m a t o g r a p h y of p r o i n h i b i t o r on CM-Sephadex. F r e s h s u p e r n a t e (o. 5 ml) was applied to a 25 cm × I . I cm CM S e p h a d e x c o l u m n equilibrated with Buffer B and developed w i t h the same buffer until F r a c t i o n IO, when elution of hemoglobin was initiated b y addition of i.o M KCI in Buffer B. The flow rate was 2o ml/h, and 1.9-ml fractions were collected. I n h i b i t o r activity was determined in duplicate. Fig. 7. Effect of h e m i n concentration on suppression of inhibitor formation. CMS-proinhibitor, p r e p a r e d as described in Methods and r e c o n s t i t u t e d to five times its concentration in the original supernate, was w a r m e d at 34 °C for 4 h in the presence of the indicated concentrations of hemin. The p l o t t e d p o i n t s represent duplicate d e t e r m i n a t i o n s of the inhibitor formed, expressed as units/ml, a d j u s t e d to its c o n c e n t r a t i o n in the original supernate. The inhibitory activity of the u n i n c u b a t e d sample (5 ° units/ml) h a s been subtracted. Taking the proinhibitor concentration in the original s u p e r n a t e as i, p r o i n h i b i t o r concentrations were: 5 ( • - • ) ; 2.5 ( O O ) ; I ( & A ) ;
0.5 (rq-E3); 0.25 (A-A). The effect of heroin on inhibitor tormation by the CMS-proinhibitor was studied as a function of both hemin and proinhibitor concentrations. The results indicate that as the proinhibitor was diluted, a correspondingly lower concentration of hemin was capable of producing the same degree of suppression of inhibitor formation (Fig. 7)Thus, a given ratio ot hemin to proinhibitor (Fig. 7, inset) produced the same suppression at all the proinhibitor concentrations tested. These data also indicate that the reaction in which inhibitor is formed is relatively insensitive to dilution. A similar pattern of hemin suppression of inhibitor formation was observed with fresh supernate (Table I). When the supernate was diluted by a factor of 5, Biochim. Biophys. Acta, 287 (I972) 340-352
347
INHIBITOR OF GLOBIN CHAIN INITIATION TABLE I SUPPRESSION
OF
INHIBITOR
FORMATION
AT
DIFFERENT
CONCENTRATIONS
OF
SUPERNATE
AND
HEMIN
Fresh supernate at the indicated concentrations (that of undiluted supernate is taken as I) was incubated with the indicated levels of hemin at 34 °C for 4 h, and then inhibitor formation was determined at 3 different doses of each sample. In (a), hemin was added to the supernate and the total diluted, whereas in (b), diluted hemin was added to the diluted supernate. All dilutions were with water, and units/ml refers to inhibitor adjusted to its concentration in the original supernate.
Concentration o/ supernate
Concentration of heroin (#M)
Inhibitor/ormation (units /ml)
i I I I/5 I/5 1/5 t/5
-7 35 7 (a) 7 (b) 35 --
780 535 98 IOI lO 5 142 590
only one-fifth the hemin concentration was required to produce the same suppression of i n h i b i t o r f o r m a t i o n . I n a d d i t i o n , t h e t o t a l a m o u n t of i n h i b i t o r f o r m e d i n t h e d i l u t e d s u p e r n a t e w a s o n l y 25 % less t h a n t h a t f o r m e d in t h e o r i g i n a l a m o u n t of u n d i l u t e d supernate. W h e n t h e s e s t u d i e s w e r e e x t e n d e d t o t h e h e r o i n s t i m u l a t i o n of i n c o r p o r a t i o n i n t h e r e t i c u l o c y t e cell-free s y s t e m , t h e s a m e d e p e n d e n c e w a s o b s e r v e d (Fig. 8). A s t h e c o n c e n t r a t i o n of l y s a t e i n t h e s y s t e m w a s r e d u c e d b y d i l u t i o n w i t h w a t e r , t h e r e was a parallel decrease in the optimum hernin concentration. When the lysate was diluted with fresh supernate, the hemin optimum remained the same. This indicated t h a t i t w a s t h e r a t i o of h e r o i n t o s o m e c o m p o n e n t i n t h e s u p e r n a t e , p r e s u m a b l y t h e proinhibitor, which was critical.
81
I
I
~f
I
I-
o
5
io
I
I
I
I
I
.|
+40~1 15
zo
H20
z5
HEMIN CONCENTRATION (I.LM)
-
3o
Fig. 8. Hemin optimum for globin synthesis in cell-free systems containing different proportions of lysate. The indicated proportions of lysate, supernate, and water were incubated with 3 °/~1 of master mix and 30/,I of water a t 34 °C for 90 min. The indicated hemin concentration was t h a t in the final incubation volume, I IO/[gl. Leucine incorporation represents hot trichloroacetic acidprecipitable radioactivity per I 1.4/zl of lysate. All incubations were performed in duplicate.
Biochim. Biophys. Acta, 287 (1972) 340-352
348
M. GROSS, M. RABINOVITZ
Rapid/ormation o/inhibitor induced by sullhydryl reagents Sulfhydryl reagents promoted a rapid formation and subsequent rapid inactivation of an inhibitory activity when incubated with the CMS-proinhibitor fraction and then neutralized with dithiothreitol (Fig. 9). When N-ethylmaleinfide or o-iodosobenzoate was mixed with the CMS-proinhibitor fraction during the course of the formation of the natural inhibitor, there was a rapid formation of additional inhibitory activity. This activity reached a maximal level of about 18oo units/ml at every time tested, the same level reached when the proinhibitor was completely converted to the inhibitor by warming alone (Fig. 9). Thus, as the amount of proinhibitor was depleted with its conversion to natural inhibitor, progressively less additional inhibitory activity could be induced by N-ethylmaleimide or o-iodosobenzoate. When CMS-proinhibitor had been fully converted to inhibitor by warming for 14 h at 34 °C, the addition of N-ethylmaleimide or o-iodosobenzoate produced no I00~
!
-
,
°o
6[)21
•
] 4[)
18
b
g
15
•
~ s
/ z
hibito¢
NEM-I nhlbdOr
.~I~.~-
o
~4~_~_~e.-~ ~ - ~
Standard Inhibitor 7 ~
~'o~'~ 6
%
~ 8 HOURS
I0
12
14
05
I'0 [15 [NHIB4TOR (/~1)
20
Fig. 9. Effect of sulfhydryl reagents on inhibitor formation. CMS-proinhibitor, prepared as indicated in Methods and at 3 times its c o n c e n t r a t i o n in the original supernate, was w a r m e d at 34 °C in the presence ( 0 - 0 ) and absence ( O - O ) of I 2 o / , M hemin. At the indicated times, 25-~1 aliquots of the sample i n c u b a t e d w i t h o u t h e m i n were mixed w i t h 25 ~ul of i.o • lO -3 M Nethylmaleimide ( a - / x ) or 2.5 • lO -3 M o-iodosobenzoate ([:]-[3), b o t h p r e p a r e d in IO times conc e n t r a t e d Buffer A. io-~ul aliquots of these m i x t u r e s were t h e n neutralized w i t h 5 #1 of o. 5 • lO -3 M or 2. 5 • lO -3 M dithiothreitol in IO times c o n c e n t r a t e d Buffer A, respectively, after 2, 6 and 15 rain. I n c u b a t i o n was c o n t i n u e d at 34 °C for 8 m o r e m i n u t e s before a s s a y i n g inhibitory a c t i v i t y w h i c h is expressed as u n i t s / m l of the original supernate. Neutralized N-ethylmaleimide, o-iodosobenzoate or dithiothreitol alone, at the levels used for e s t i m a t i o n of inhibitory activity had no effect on the a s s a y system. Fig. io. Dose dependence of inhibition p r o d u c e d b y o-iodosobenzoate-(OIB), N-ethylmaleimide(NEM), and s t a n d a r d inhibitor, o-Iodosobenzoate- a n d N - e t h y l m a l e i m i d e - i n h i b i t o r s were p r e p a r e d as described in Methods. S t a n d a r d i n h i b i t o r represents i n h i b i t o r y s u p e r n a t e which h a d been w a r m e d w i t h o u t h e m i n for 14 h at 37 °C and t h e n partially purified on CM-Sephadex in a m a n n e r identical to t h e p r e p a r a t i o n of p r o i n h i b i t o r (Methods). I t was t h e n lyophilized and dissolved in a v o l u m e of w a t e r e q u i v a l e n t to the v o l u m e of the original supernate.
Biochim. Biophys. Acid, 287 (I972) 340-352
INHIBITOR
OF
GLOBIN
CHAIN
349
INITIATION
more inhibitory activity. In addition, if CMS-proinhibitor was mixed with the sulfhydryl reagents and the reaction terminated with dithiothreitol within 15 min, further warming of this mixture produced no additional inhibitory activity. When CMSproinhibitor was incubated either with dithiothreitol or with the sulfhydryl reagents neutralized with dithiothreitol, the rate of formation of inhibitory activity was the same as that when CMS-proinhibitor was warmed alone, (data not shown). These results suggest that N-ethylmaleimide or o-iodosobenzoate, when added to CMSproinhibitor, rapidly convert the proinhibitor to a component which is similar to the natural inhibitor. When the dose of inhibitor, formed b y the action of N-ethylmaleimide or o-iodosobenzoate on CMS-proinhibitor, was varied, the degree of inhibition of cell-free hemoglobin synthesis followed the same semi-log relationship as that observed with 16 ~
]
~
--
I
T - - ]
I o.7
I
I
I
'°i
i
i 1
40 Vo I
1 1/
0.5
I
/
; ,~| ~ !?
(Hemoglobin
1
-
elutio, at 516ml)
I0 i30 ~
~'0.4
v B-
20 ~
0.3
i C~2
i 0.1
l
Ij~.%;:..~;" o
250
i
, "%.. ~2s
300 350 ELUTION VOLUME (ral)
400 MINUTES
F i g . i I. C h r o m a t o g r a p h y o n S e p h a d e x G - 2 o o of p r e c u r s o r for n a t u r a l i n h i b i t o r a n d t h a t i n d u c i b l e b y s u l f h y d r y l r e a g e n t s . A 145 c m x 2.5 c m S e p h a d e x G - 2 o o c o l u m n w a s p r e p a r e d a n d o p e r a t e d as i n d i c a t e d i n M e t h o d s . F r e s h s u p e r n a t e (4 m l ) w a s a p p l i e d , t h e c o l u m n d e v e l o p e d w i t h B u f f e r A, a n d 4 - m l f r a c t i o n s w e r e c o l l e c t e d . A f t e r d e t e r m i n a t i o n of t h e a b s o r b a n c e a t 28o n m , all f r a c t i o n s w e r e l y o p h i l i z e d a n d r e d i s s o l v e d i n 0.4o m l of w a t e r . I n h i b i t o r y a c t i v i t y w a s d e t e r m i n e d i n e a c h f r a c t i o n : w i t h o u t f u r t h e r t r e a t m e n t ( • - • • • ) ; a f t e r w a r m i n g a l i q u o t s a t 34 °C for 4 h ( O - - - O ) ; a n d a f t e r m i x i n g I O # 1 a l i q u o t s of e a c h f r a c t i o n w i t h IO p l of I . O . lO -2 M N - e t h y l m a l e i m i d e (zX- • - A ) o r 2. 5 • lO _2 M o - i o d o s o b e n z o a t e (VI- - -[~) i n IO t i m e s c o n c e n t r a t e d B u f f e r A, w a r m i n g for 2 m i n a t 34 °C a n d t h e n n e u t r a l i z i n g b y i n c u b a t i n g for a n a d d i t i o n a l 15 r a i n w i t h IO y1 of o. 5 • i o - s or 2.5 • lO -2 M d i t h i o t h r e i t o l , i n IO t i m e s c o n c e n t r a t e d B u f f e r A, r e s p e c t i v e l y . I n h i b i t o r y a c t i v i t y is e x p r e s s e d a s t h a t i n t h e o r i g i n a l , u n c o n c e n t r a t e d e l u a t e . F i g . 12. E f f e c t of h e r o i n o n i n h i b i t o r f o r m a t i o n b y s u l f h y d r y l r e a g e n t s . C M S - p r o i n h i b i t o r , i n o n e - t h i r d t h e v o l u m e of t h e o r i g i n a l s u p e r n a t e , w a s m i x e d w i t h a n e q u a l v o l u m e of i . o • I o -2 M N - e t h y l m a l e i m i d e a t o °C ( O , O ) , 2.5 • i o - I M o - i o d o s o b e n z o a t e a t 25 °C ( A , • ) , o r 2.0 • lO -2 M N - e t h y l m a l e i m i d e a t 34 °C (F1, It), a l l p r e p a r e d i n IO t i m e s c o n c e n t r a t e d B u f f e r A, i n t h e p r e s e n c e (closed s y m b o l s ) a n d a b s e n c e ( o p e n s y m b o l s ) of h e m i n , f i n a l c o n c e n t r a t i o n , 60 p M . A t t h e i n d i c a t e d t i m e s , io-~ul a l i q u o t s of e a c h r e a c t i o n m i x t u r e w e r e n e u t r a l i z e d w i t h 5/~1 of d i t h i o t h r e i t o l p r e p a r e d i n IO t i m e s c o n c e n t r a t e d B u f f e r A, a t c o n c e n t r a t i o n s of 0. 5 • lO -2 M, 2.5 • lO -2 M, a n d i . o • i o -~ M, r e s p e c t i v e l y , w i t h a d d i t i o n a l i n c u b a t i o n a t 34 °C for 15 m i n . I n h i b i t o r y a c t i v i t y was assayed in duplicate.
Biochim. Biophys. Acta, 287 ( I 9 7 2 ) 3 4 0 - 3 5 2
35 °
M. GROSS, M. RABINOVITZ
natural inhibitor (Fig. 1o). The component which becomes inhibitory when mixed with N-ethylmaleimide or o-iodosobenzoate co-chromatographs on Sephadex G-2oo with the natural proinhibitor (Fig. 1i). Heroin, however, only slightly retarded the formation of inhibitory activity when CMS-proinhibitor was incubated with either sulfhydryl reagent (Fig. 12). Several additional observations suggest that the inhibitory component, produced by the action of N-ethylmaleimide or o-iodosobenzoate on CMS-proinhibitor, m a y have the same mode of action as natural inhibitor. The inhibitory action of the former and that of the latter was prevented by a partially purified, 0.55 M KC1 wash of reticulocyte ribosomes 1~, I-fraction 14,2~ (Fig. 13). In addition, the inhibitor pro-
II I
I0 "
i
I
~0""~0~0 ~
I
I
I
O.
9I
g~
20 0 40 I-FACTOR {~g)
~o
60
Fig. 13. E f f e c t of p a r t i a l l y p u r i f i e d I - f a c t o r on t h e i n h i b i t i o n b y n a t u r a l , N - e t h y l m a l e i m i d e , a n d o - i o d o s o b e n z o a t e - i n h i b i t o r s . Cell-free s y s t e m s c o n t a i n e d t h e i n d i c a t e d a m o u n t s of I - f a c t o r a n d a p p r o x i m a t e l y I u n i t of n a t u r a l ( O - - - O ), N - e t h y l m a l e i m i d e - ([2]- - -[2] ), or o - i o d o s o b e n z o a t e ( A- - - A ) i n h i b i t o r in 3 ° / z l ; 3 °/~1 of m a s t e r m i x ; 5 ° / , 1 of l y s a t e a n d a fi na l he roi n c o n c e n t r a t i o n of 20/~M. C o n t r o l s a m p l e s ( 0 - - - 0 ) r e c e i v e d no a d d e d i n h i b i t o r . T h e p u r i f i e d I - f a c t o r 18 w a s a o.55 M KC1 e x t r a c t of r e t i c u l o c y t e ribosomes, d i l u t e d to o.I M KC1 w i t h buffe r (Tris-HC1, i o mM p H 7.5, d i t h i o t h r e i t o l , I raM; a n d o . i mM E D T A ) . The d i l u t e d e x t r a c t w a s c o n c e n t r a t e d b y u l t r a f i l t r a t i o n a n d a p p l i e d to a D E A E - c e l l u l o s e c o l u m n e q u i l i b r a t e d w i t h o. i M KC1 in t h e a b o v e buffer. A f t e r w a s h i n g t h e c o l u m n w i t h o.i M IZC1 in buffe r t o r e m o v e h e m o g l o b i n , i t w a s f u r t h e r w a s h e d , a n d t h e n t h e I - f a c t o r w a s e l u t e d w i t h o.16 M KC1 in buffer. I n c u b a t i o n s w e re c a r r i e d o u t a t 34 °C for 90 m i n a n d t h e h o t t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e r a d i o a c t i v i t y w a s d e t e r m i n e d in 25-/zl a l i q u o t s . N o t e t h e b r e a k in scale of t h e o r d i n a t e .
duced by N-ethylmaleimide action on CMS-proinhibitor caused complete disaggregation of polyribosomes within 5 min when incubated with lysate and an energy source at 34 °C, but had no effect in the absence of an energy source (Fig. 14). Natural inhibitor had an identical action on the polyribosomes. In neither case can the inhibitory activity be attributed to ribonuclease, which degrades the polyribosomes in the absence of an energy source. Biochim. Biophys. Acta, 287 (1972) 340-352
INHIBITOR OF GLOBIN CHAIN INITIATION
0.6[ A
°41/
351
F
c
i E
A2~oOB8 06I 02
CMFROMMENISCUS
Fig. 14. Effect of n a t u r a l a n d N - e t h y l m a l e i m i d e - i n d u c e d i n h i b i t o r s on t h e p o l y r i b o s o m e profile of r e t i c u l o c y t e lysates. Cell-free s y s t e m s , c o n t a i n i n g 15o/~1 of lysate, 9o/~1 of w a t e r (A, C, E) or m a s t e r m i x (B, D, F), a n d 15 i n h i b i t o r u n i t s of N - e t h y l m a l e i m i d e - i n h i b i t o r (C a n d D) or of n a t u r a l i n h i b i t o r (E a n d F) in 90/~1 of Buffer A, or 90/~1 of B u f f e r A alone (A a n d B), were i n c u b a t e d w i t h 2o/~M h e m i n a t 34 °C for 5 min. All s a m p l e s i m m e d i a t e l y received 300/~1 of 2-deoxyglucose, o . i o M, c o n t a i n i n g 15o u n i t s of h e x o k i n a s e 18, a n d were q u i c k l y cooled to o °C. 5oo~1 of each s a m p l e were l a y e r e d on a linear I5 to 3 ° % sucrose gradient. A f t e r c e n t r i f u g a t i o n a t 41 ooo rev./ m i n for lO 5 m i n in t h e S W 41 rotor, t h e c o n t e n t s of t h e t u b e s were p u m p e d b y u p w a r d d i s p l a c e m e n t t h r o u g h a flow cell in a B e c k m a n D U s p e c t r o p h o t o m e t e r , a n d t h e elution profile a t 26o n m w a s m o n i t o r e d w i t h a Gilford recorder.
Also tested was the possibility that other compounds which could influence the conformation of proteins would cause the rapid formation of inhibitory activity when added to CMS-proinhibitor. Treatment with 8 M urea, 5 M guanidine -HC1, or precipitation at p H 5 were all ineffective in promoting the rapid formation of inhibitory activity, (data not shown). These results suggest that some type of sulfhydryl reaction m a y be involved in the natural conversion of proinhibitor to inhibitor when supernate is warmed without heniin.
DISCUSSION
The complete conversion of proinhibitor to inhibitor, which normally requires incubation at 34 °C for 12 to 15 h, is rapidly accelerated b y such sulfhydryl reagents as N-ethylmaleimide and o-iodosobenzoate. By a number of criteria, the component upon which the sulfhydryl reagents act seems identical to the proinhibitor ,and the inhibitor whose formation they induce blocks globin chain initiation as does the natural inhibitor. The reactions occurring during the formation of natural inhibitor are still unknown, but our observations suggest that the change from proinhibitor to inhibitor m a y be the result of specitic conformational changes. First, the rate of the natural reaction is reduced b y less than a factor of 2 when the system which forms inhibitor is diluted b y a factor of 2o. Second, partial purification of the inhibitor fornfing activity b y ion exchange and gel filtration suggests that it m a y be one component, the proinhibitor. Therefore, we suggest that the reticulocyte contains some protein which, in the absence of hemin and at physiological temperatures, undergoes an alteration in conformation possibly involving disulfide bond formation 6r interchange, and becomes an inhibitor of globin chain initiation. In the course of this change, the inhibitory site on the proinhibitor m a y become unmasked. Biochim. Biophys. Acta, 287 (1972) 340-352
352
M. GROSS, M. RABINOVITZ
K o s o w e r et al. 2~ h a v e f o u n d t h a t o x i d i z e d g l u t a t h i o n e will i n h i b i t g l o b i n c h a i n i n i t i a t i o n i n a r a b b i t r e t i c u l o c y t e cell-free s y s t e m s u p p l e m e n t e d w i t h h e r o i n . W e t e s t e d w h e t h e r o x i d i z e d g l u t a t h i o n e w o u l d i n d u c e t h e f o r m a t i o n of i n h i b i t o r y a c t i v i t y w h e n i n c u b a t e d w i t h f r e s h s u p e r n a t e i n t h e p r e s e n c e of h e m i m T h e o x i d i z e d g l u t a t h i o n e w a s t h e n r e d u c e d w i t h d i t h i o t h r e i t o l . A c o n c e n t r a t i o n of g l u t a t h i o n e d i s u l f i d e , I 0 -a M, w h i c h i n h i b i t e d cell-free g l o b i n s y n t h e s i s m a x i m a l l y , g e n e r a t e d n o d e t e c t a b l e inhibitory activity in the supernate in 5 rain. Thus the inhibition by oxidized glutat h i o n e a p p e a r s t o b e m e d i a t e d b y a n o t h e r m e c h a n i s m . I t is of i n t e r e s t t h a t cell-free p r o t e i n s y n t h e s i s b y m i c r o s o m e s f r o m n o r m a l a n d r e g e n e r a t i n g l i v e r is also a f f e c t e d b y o x i d i z e d a n d r e d u c e d s u l f h y d r y l r e a g e n t s 26. T h e r e l a t i o n s h i p b e t w e e n t h e s e f i n d i n g s a n d t h o s e w i t h r e t i c u l o c y t e s will r e q u i r e f u r t h e r i n v e s t i g a t i o n .
REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 ':, 26
J. Kruh and H. Borsook, J. Biol. Chem., 220 (1956) 9o5. G. P. Bruns and I. M. London, Biochem. Biophys. Res. Commun., 18 (1965) 236. H. S. W a x m a n and M. Rabinovitz, Biochem. Biophys. Res. Commun., 19 (1965) 538. A. I. Grayzel, P. H6rchner and I. M. London, Proc. Natl. Acad. Sci. U.S., 55 (1966) 650. H. S. W a x m a n and M. Rabinovitz, Biochim. Biophys. Acta, 129 (1966) 369. H. S. Waxman, M. L. Freedman and M. Rabinovitz, Bioehim. Biophys. Acta, 145 (1967) 353. S. D. Adamson, E. Herbert and W. Godchaux, Arch. Bioehem. Biophys., 125 (1968) 671. W. V. Zucker and H. M. Schulman, Proc. Natl. Acad. Sci. U.S., 59 (1968) 582. S. D. Adamson, E. Herbert and S. F. Kemp, J. Mol. Biol., 42 (1969) 247. C. R. Maxwell and M. Rabinovitz, Biochem. Biophys. Res. Commun., 35 (1969) 79. M. Rabinovitz, M. L. Freedman, J. M. Fisher and C. R. Maxwell, Cold Spring Harbor Syrup. Quant. Biol., 34 (1969) 567. G. A. Howard, S. D. Adamson and E. Herbert, Biochim. Biophys. Acta, 213 (197 o) 237. C. R. Maxwell, C. S. Kamper and M. Rabinovitz, J. Mol. Biol., 58 (1971) 317 S. D. Adamson, P. M. Yau and E. Herbert, J. Mol. Biol., 63 (1972) 247. M. Gross and M. Rabinovitz, Proc. Natl. Acad. Sci. U.S., 69 (1972) 1565. M. O. Dayhoff and R. V. Eck, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Silver Spring, Md., 1967, 1968. C. R. Maxwell and C. Mencken, Anal. Biochem., 44 (1971) 235. S. Mizuno, J. M. Fisher and M. Rabinovitz, Bioehim. Biophys. Acta, 272 (1972) 638. W. V. Zucker and H. M. Schulman, Bioehim. Biophys. Aeta, 138 (1967) 400. A. S. Tavill, A. I. Grayzel, I. M. London, M. K. Williams and G. A. Vanderhoff, J. Biol. Chem., 243 (1968) 4987 . A. S. Tavill, G. A. Vanderhoff and I. M. London, J. Biol. Chem., 247 (1972) 326. H. M. Dintzis, Proc. Natl. Acad. Sci. U.S., 47 (1961) 247. M. Rabinovitz, and J. M. Fisher, Biochim. Biophys. Acta, 91 (1964) 313 . R. L. Miller and R. Schweet, Arch. Biochem. Biophys., 125 (1968) 632. N. S. Kosower, G. A. Vanderhoff, B. Benerofe, T. H u n t and E. M. Kosower, Biochem. Biophys. Res. Commun., 45 (1971 ) 816. R. D. Nolan and M. B. Hoagland, Biochim. Biophys. Acta, 247 (1971) 609.
Biochim. Biophys. Acta, 287 (1972) 34o-352
BIOCHIMICA ET BIOPHYSICA ACTA
8BA 97465
CONTROL OF GLOBIN S Y N T H E S I S BY H E M I N : FACTORS I N F L U E N C I N G FORMATION OF AN I N H I B I T O R OF G L O B I N CHAIN I N I T I A T I O N IN R E T I C U L O C Y T E LYSATES M A R T I N G R O S S " AND M A R C O R A B I N O V I T Z
Laboratory of Physiology, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.) ( R e c e i v e d J u n e i 2 t h , 1972)
S U MMARY
The control of globin synthesis by hemin in rabbit reticulocyte lysates is mediated by the formation of an inhibitor of globin chain initiation. This cytoplasmic repressor does not appear to be a product derived from newly synthesized globin. It is formed in the absence of hemin from a protein in the post-ribosomal fraction without participation of free, low molecular weight components. This proinhibitor and the inhibitor are eluted at the same point on a Sephadex G-2oo column, which corresponds to a molecular weight of 3.0 • lO 5. Although the complete transition of proinhibitor to inhibitor in the ribosome-free supernatant fraction takes place over a period of 12 to 15 h, partially purified proinhibitor can be converted to inhibitor within 15 min b y reaction with N-ethylmaleimide or o-iodosobenzoate. These findings suggest that inhibitor formation m a y involve a specific conformational change in the proinhibitor molecule.
INTRODUCTION
The maintenance of globin synthesis in the reticulocyte is dependent upon the availability of hemin 1'2. In the absence of iron for heroin synthesis 3 or of heroin itself 3-5, the cellular polyribosomes disaggregate, indicating that control is at the site of chain initiation e. Reticulocyte lysates are similarly dependent upon heroin for sustained globin synthesis 7-9. This regulation is mediated through the suppression by heroin of formation of an inhibitor of globin chain initiation in the post-ribosomal supernatant fraction 1°-15. Within 30 rain of its formation, this inhibitor, a translational repressor, can be completely inactivated by heroin and is denoted as reversible inhibitor ~5. With longer incubations in the absence of heroin, the inhibitory activity can no longer be overcome by hemin and the material formed is denoted as irreversible inhibitor 10-15. In this report we describe experiments designed to study the nature of the precursor of the inhibitor, termed proinhibitor, the protective effect of heroin, and the reaction involving transformation of proinhibitor to inhibitor. The formation of irreversible inhibitor was followed since it can be quantitatively assayed and appears to be the ultimate product of the reactions being studied. A b b r e v i a t i o n : H E P E S , N-e-hydroxyethylpiperazine-N'-2-ethanesulfoniatc. * P r e s e n t a d d r e s s : D e p a r t m e n t of P a t h o l o g y , U n i v e r s i t y of Chicago, Chicago, Ill., U.S.A.
Biochim. Biophys. Acta, 287 (i972) 34o-352
INHIBITOR OF GLOBIN CHAIN INITIATION
34 [
METHODS
Sources o/materials The sources of materials required for the cell-free system were indicated previously n,13. N-Ethylmaleimide and o-iodosobenzoate were purchased from Sigma, and dithiothreitol, from CalBiochem. H E P E S (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid) and horse spleen apoferritin were from CalBiochem, and pyruvate kinase, from Worthington. Sephadex G-25, G-5o, and G-2oo, blue dextran 2000, and CM-Sephadex C-5o were from Pharmacia, and Bio-Gel P-I5O, from Bio-Rad. Aquasol was purchased from New England Nuclear.
Cell-/ree incubations and assay/or inhibitor The method of preparation of reticulocyte lysate from phenylhydrazine-treated rabbits has been described 18. Incubation mixtures contained 5 vol. of lysate, 3 vol. of master mix, and 3 vol. of water or of a solution of the material being studied. Unless otherwise indicated, heroin was added so that the final concentration was 20/*M. The assay of the inhibitor was carried out as previously describedlS; one unit of inhibitor being defined as that amount which reduces to 5 ° % the stimulation of protein synthesis by hemin in a standard incubation system. Master mix contained the following components in amounts to yield the indicated final concentration in the total reaction mixture: KC1 (75 mM); MgC1, (2 nlM); ATP (0.5 mM); GTP (0.2 raM); creatine phosphate (15 raM); creatine phosphokinase (45 units/ml); L-[14C]leucine (0.3 mM, 5/*Ci/#mole) and the other 19 amino acids at concentrations corresponding to the amino acid composition of rabbit hemoglobin 16. Unless otherwise indicated, incubations were carried out at 34 °C for 9 ° rain, and the hot trichloroacetic acidinsoluble radioactivity in 25-/.1 aliquots of the total reaction mixture was determined by plating on Millipore filters and counting in a gas-flow counter with Micromil window. The post-ribosomal supernatant fraction (denoted supernate) was prepared by centrifuging lysate in cut-down tubes at approximately 400 ooo × g for 60 min in the SW-56 rotor 17.
Chromatography o/the products o/the cell-]ree reaction on Biogel P-z5o All preparative and chromatographic procedures were performed at o to 4 °C unless otherwise indicated. The post-ribosomal fraction, (3-3 ml), prepared from the total reaction mixture by centrifugation at 3oo ooo × g (average) for 2 h in the SW56 rotor, was applied to the top of a 145 cm × 2.5 cm Biogel P-I5O column equilibrated with 3.7 mM H E P E S (pH 7.2) and o.o275 M KC1 (Buffer A) and eluted under gravity flow with this buffer. The flow rate was 15 ml/h, and 3.5-ml fractions were collected. The radioactivity in fractions not containing hemoglobin was determined on o.5o-ml aliquots in IO ml Aquasol with a Packard model No. 4322 liquid scintillation spectrometer (efficiency 9 ° %). Diluted aliquots of the fractions containing hemoglobin were evaporated to dryness on planchets and counted in a Nuclear Chicago gas-flow counter with Mieromil window (efficiency 34 %). Radioactivity determined by the latter method was corrected to 9 ° % efficiency to permit direct comparison with fractions counted on the scintillation spectrometer.
Preparation o/ proinhibitor on CM-Sephadex Large-scale preparation of proinhibitor was accomplished by applying 20 ml Biochim. Biophys. Acta, 287 (i972) 34o-352
342
M. GROSS, M. RABINOVITZ
of fresh supernate to a 4 ° cm × 4.5 cm CM-Sephadex colunm equilibrated with 3.7 mM H E P E S , pH 7.2 (Buffer B). Elution was with Buffer B, and the region capable of forming inhibitor, which corresponds to the void volume of the column, was pooled and lyophilized. The lyophilized product was dissolved in a volume of water equivalent to one-third or one-fifth the volume of the original supernate and stored at --8o °C. It is denoted as CMS-proinhibitor.
Determination o/molecular weight o/ proinhibitor and o/inhibitor on a Sephadex G-2oo column A 14o cm × 2.5 cm Sephadex G-2oo column, equilibrated with Buffer A, was developed with this buffer at a rate of 16 ml/h. The indicated markers were each applied separately to the column in 4.0 ml, fractions containing 4.0 ml were collected, and the elution volume was determined by the peak of absorbance at 280 nm (apoferritin and pyruvate kinase), 260 nm (blue dextran), or 415 nm (rabbit hemoglobin). The location of inhibitor was determined as the peak of activity after the application of I ml of inhibitory supernate (3000 units) mixed with 3 ml of water. To locate proinhibitor, 4.0 ml of fresh supernate were applied to the column and the capacity to form inhibitor was determined by measuring inhibitory activity after warming each column fraction at 34 °C for 4 h.
Preparation o] inhibitory activity/rom CMS-proinhibitor with sul/hydryl reagents I vol. of CMS-proinhibitor, concentrated to one-third the volume of the original supernate, was mixed with i vol. of either IO mM N-ethylmaleimide or 25 mM o-iodosobenzoate, both prepared in IO times concentrated Buffer A. After incubation for 5 min at 34 °C, the N-ethylmaleimide in the treated sample was neutralized with I vol. of 5 mM dithiothreitol in IO times concentrated Buffer A. After 2 min incubation at 34 °C, the o-iodosobenzoate in the treated sample was neutralized with i vol. of 25 mM dithiothreitol. After incubation of both samples at 34 °C for a total of 15 min, they were cooled to o °C and used immediately. Inhibitor formed by incubation of CMS-proinhibitor with N-ethylmaleimide and o-iodosobenzoate is denoted as N-ethylmaleimide-inhibitor and o-iodosobenzoate-inhibitor, respectively.
RESULTS
The inhibitor is not derived/rom newly/ormed globin The inhibitor which appears when post-ribosomal supernate of a rabbit reticulocyte lysate is warmed without heroin, is a protein 14,1s which is derived from a preformed proinhibitor 1~. Since reticulocytes contain a pool of free :c-chains which are unstable in the absence of hemin 19-*t, we tested whether the proinhibitor was a form of globin or was derived from globin. Taking advantage of the fact that there is a long lag in the formation of inhibitor at 25 °C (ref. I3), proteins of a lysate were labeled with [~*Cjleucine in the absence of hemin at this temperature. The products of the reaction, freed of ribosomes, were analyzed on a Biogel P-I50 column either immediately (Fig. I) or after warming at 34 °C (Fig. 2). In the former, a small amount of radioactivity and inhibitory activity was eluted with the void volume. About onehalf of the remaining radioactivity was coincident with hemoglobin, and the other
Biochirn. Biophys. Acta, 287 (1972) 340-352
343
INHIBITOR OF GLOBIN CHAIN INITIATION ~
90
~a
.~
A
{6o 75
5
06
5
I
i\
cz:o
-
\
f~'
0.2~
(n
.... 77 50
60
70 FRACTION NUMBER
80
90
I00
I10
Fig. I. Gel filtration of s u p e r n a t e containing proinhibitor and labeled globin prior to the formation of inhibitor. An incubation mixture, containing 3.o ml of lysate w i t h o u t added hemin, 1.8 ml of m a s t e r mix and 1.8 ml of water, was incubated a t 25 °C for 60 min. The ribosomes were t h e n r e m o v e d b y centrifugation, the s u p e r n a t e fractionated on a Biogel P - i 5 o c o l u m n and the inhib i t o r y p o t e n c y a n d r a d i o a c t i v i t y of fractions were determined as described in Methods. The abscissa has been divided to p e r m i t a change in scale for b o t h radioactivity and absorbance.
0
?-
9O
&
25
! i
o
_o
i .
c
~ 5o
~o
it
60
il
75
o
0.6
g 2m~~ 2 §.~
O
Z i
02 i
~5
o
g J I
50
60
70
80
90
I00
IiO
FRACTION NUMBER
Fig. 2. Gel filtration of s u p e r n a t e containing p r o i n h i b i t o r and labeled globin after f o r m a t i o n of inhibitor. After the r e m o v a l of ribosomes, the s u p e r n a t e was incubated at 34 °C for 16 h before fractionation on a Biogel P-I5O column. Conditions are as indicated in the legend to Fig. I.
half was eluted at a position corresponding to a molecular weight of about 33 ooo. The latter component was mixed with unlabeled rabbit hemoglobin, hemin was split from globin with acid-acetone, and ~- and fl-chains were separated by carboxymethyl cellulose chromatography 2z,23. Under these conditions 95 % of the labeled protein co-chromatographed with the ~-chain. When the products of the cell-free reaction were separated on Biogel P-I5O after warming at 34 °C (Fig. 2), additional inhibitory Biochim. Biophys. Acta, 287 (1972) 340-352
344
M. GROSS, M. RABINOVITZ
activity was eluted with the void volume, but the amount of radioactivity in this region decreased as a result of the incubation. The labeled ~-globin fraction was found in a component which was eluted with hemoglobin and was not in the inhibitor region. The results suggest that the inhibitor is not derived from newly synthesized 0~-globin.
The molecular size o/the components o/ the inhibitory/orming system To determine whether any low molecular weight component is required for inhibitor formation, fresh, ribosome-free supernate was chromatographed on Sephadex G-25 and G-5o columns, and the fraction which was eluted at the void volume was warmed to determine its ability to form inhibitor. In the course of these studies, we found that the eluate from Sephadex G-25 columns was inhibitory before it was warmed. If, however, such a column was pretreated by an initial passage of the super-
8~
A
I
£-
i
200
40 4-
i
~5o
I|
I 12~
i
B
~c ~z
2C o
n~
N
9
~m Io 5=
o
1
m~
5O
2 I
2
5
4
5
6
7
HOURS
I 40
50
FRACTION NUMBER
Fig. 3. Inhibitor-forming capacity of the void volume eluate from supernates filtered on Scphadex G-25, Sephadex G-5 o, and Biogel P-I5O columns. (A) Fresh supernate (ioo/~1) was applied to i8 cm × 0.6 cm Sephadex G-25 ([], It) and Sephadex G-5o (&, A) columns, equilibrated with Buffer A. The Sephadex G-25 column h a d been pretreated with 2o0/~1 of fresh, normal rabbit hemolysate a n d t h e n washed t h o r o u g h l y with Buffer A. E a c h column was developed with Buffer A, and the void volume fractions, containing hemoglobin as a marker, were pooled. These eluates and an aliquot of the original s u p e r n a t e (O, O) were warmed at 34 °C for the indicated times in the presence (closed symbols) and absence (open symbols) of 35/2M hemin. Inhibitor activity was m e a s u r e d in duplicate samples, a n d the results are expressed as units/ml corrected to the concentration in the original supernate. (B) The pooled inhibitor-forming activity from the Biogel P-I5O column shown in Fig. 4 (Fractions 30-35) was lyophilized and t h e n reconstituted with water to the concentration of the original supernate. Aliquots of this P-I5O fraction (A, A) a n d of the original s u p e r n a t e (O, • ) were t h e n warmed at 34 °C in the presence (closed symbols) and absence (open symbols) of 35/~M hemin. Duplicate samples were removed at the indicated times for the determination of inhibitor activity. Fig. 4. C h r o m a t o g r a p h y of proinhibitor on Biogel P-i5o. Fresh supernate (6 ml) was applied to a i oo cm × 2.5 cm Biogel P-15o column equilibrated with Buffer A, and the column was developed with Buffer A a t a rate of i i ml/h; 3.5-ml fractions were collected. Inhibitory activity of the fractions was determined in duplicate; before incubation ( & . . . &), after incubation for 4 h at 34 °C ( O - - - O ) .
Biochim. Biophys. Mcta, 287 (1972) 34o-352
INHIBITOR OF GLOBIN CHAIN INITIATION
345
nate followed by thorough washing with buffer, the void volume eluate obtained when a second aliquot of supernate was fractionated was not inhibitory. This indicated that the Sephadex G-25 contained some material, inhibitory to the reticulocyte cell-free system, which could be removed by a concentrated hemolysate solution. Consequently, columns of Sephadex G-25 were routinely treated with 200 #1 of fresh, stroma-free hemolysate from non-anemic rabbits and then washed with the elution buffer. When fresh supernate was chromatographed on either Sephadex G-25 or G-5o (Fig. 3, Frame A), the void volume eluates formed inhibitor at the same rate as the original supernate. Hemin markedly retarded this reaction in all cases. When fresh supernate was fractionated on a Biogel P-i5o column (Fig. 4), inhibitor formation, determined by warming and then assaying individual fractions for inhibitory activity, was limited to the void volume region. The rate of inhibitor formation and the degree of suppression of the reaction by hemin is the same in this Biogel P-I5O preparation as it is in fresh supernate (Fig. 3, Frame B). The results indicate that no freely dissociable, low molecular weight component is required for inhibitor formation. The molecular weights of the proinhibitor and inhibitor were estimated with a calibrated Sephadex G-2oo column (Fig. 5). Both the proinhibitor and inhibitor were eluted similarly, at an elution volume corresponding to a molecular weight of approximately 3 " lO5. This suggests that there is no detectable size change in the transformation from proinhibitor to inhibitor. Adamson et al. ~4 have obtained a molecular weight of 5 " lO5 for the inhibitor by gel chromatography on Sepharose 6B. We have also obtained a higher molecular weight estimate using Sepharose 6B in place of Sephadex G-2oo.
2~
lit
o.5 2r~
I 300
t It
I I 3,~o 400 450 ELUTION VOLUME {roll
it,, I
~
~
Fig. 5. Determination of molecular weight of proinhibitor and of inhibitor on a Sephadex G-2oo column. Experimental conditions are given in Methods. Biochim. Biophys. Acta, 287 (1972) 340-352
346
i.
GROSS, M. RABINOV1TZ
The nature o/heroin protection The proinhibitor was prepared free of hemoglobin by fractionation of the supernate on a CM-Sephadex column. It was eluted in the void volume before the low molecular weight components, while hemoglobin was adsorbed to the top of the column and could be eluted with I M KC1 (Fig. 6). The proinhibitor was concentrated by lyophilization as described in Methods and denoted CMS-proinhibitor.
T b 20
IM
Il
iCt
,o /
Az6° = 0183 A28o " 12(3
--
I00 4= 80 o~
E~$0 = 1.05 -60
A~
iN 0i
A260 = 38 /Az8°
05
O
~
115 20 FRACTION NUMBER
-
",20 i ;o t o
-
I 20
I I I ~ 40 60 80 I00 HEMIN CONCENTRATION (FM)
_y~__
200
Fig. 6. C h r o m a t o g r a p h y of p r o i n h i b i t o r on CM-Sephadex. F r e s h s u p e r n a t e (o. 5 ml) was applied to a 25 cm × I . I cm CM S e p h a d e x c o l u m n equilibrated with Buffer B and developed w i t h the same buffer until F r a c t i o n IO, when elution of hemoglobin was initiated b y addition of i.o M KCI in Buffer B. The flow rate was 2o ml/h, and 1.9-ml fractions were collected. I n h i b i t o r activity was determined in duplicate. Fig. 7. Effect of h e m i n concentration on suppression of inhibitor formation. CMS-proinhibitor, p r e p a r e d as described in Methods and r e c o n s t i t u t e d to five times its concentration in the original supernate, was w a r m e d at 34 °C for 4 h in the presence of the indicated concentrations of hemin. The p l o t t e d p o i n t s represent duplicate d e t e r m i n a t i o n s of the inhibitor formed, expressed as units/ml, a d j u s t e d to its c o n c e n t r a t i o n in the original supernate. The inhibitory activity of the u n i n c u b a t e d sample (5 ° units/ml) h a s been subtracted. Taking the proinhibitor concentration in the original s u p e r n a t e as i, p r o i n h i b i t o r concentrations were: 5 ( • - • ) ; 2.5 ( O O ) ; I ( & A ) ;
0.5 (rq-E3); 0.25 (A-A). The effect of heroin on inhibitor tormation by the CMS-proinhibitor was studied as a function of both hemin and proinhibitor concentrations. The results indicate that as the proinhibitor was diluted, a correspondingly lower concentration of hemin was capable of producing the same degree of suppression of inhibitor formation (Fig. 7)Thus, a given ratio ot hemin to proinhibitor (Fig. 7, inset) produced the same suppression at all the proinhibitor concentrations tested. These data also indicate that the reaction in which inhibitor is formed is relatively insensitive to dilution. A similar pattern of hemin suppression of inhibitor formation was observed with fresh supernate (Table I). When the supernate was diluted by a factor of 5, Biochim. Biophys. Acta, 287 (I972) 340-352
347
INHIBITOR OF GLOBIN CHAIN INITIATION TABLE I SUPPRESSION
OF
INHIBITOR
FORMATION
AT
DIFFERENT
CONCENTRATIONS
OF
SUPERNATE
AND
HEMIN
Fresh supernate at the indicated concentrations (that of undiluted supernate is taken as I) was incubated with the indicated levels of hemin at 34 °C for 4 h, and then inhibitor formation was determined at 3 different doses of each sample. In (a), hemin was added to the supernate and the total diluted, whereas in (b), diluted hemin was added to the diluted supernate. All dilutions were with water, and units/ml refers to inhibitor adjusted to its concentration in the original supernate.
Concentration o/ supernate
Concentration of heroin (#M)
Inhibitor/ormation (units /ml)
i I I I/5 I/5 1/5 t/5
-7 35 7 (a) 7 (b) 35 --
780 535 98 IOI lO 5 142 590
only one-fifth the hemin concentration was required to produce the same suppression of i n h i b i t o r f o r m a t i o n . I n a d d i t i o n , t h e t o t a l a m o u n t of i n h i b i t o r f o r m e d i n t h e d i l u t e d s u p e r n a t e w a s o n l y 25 % less t h a n t h a t f o r m e d in t h e o r i g i n a l a m o u n t of u n d i l u t e d supernate. W h e n t h e s e s t u d i e s w e r e e x t e n d e d t o t h e h e r o i n s t i m u l a t i o n of i n c o r p o r a t i o n i n t h e r e t i c u l o c y t e cell-free s y s t e m , t h e s a m e d e p e n d e n c e w a s o b s e r v e d (Fig. 8). A s t h e c o n c e n t r a t i o n of l y s a t e i n t h e s y s t e m w a s r e d u c e d b y d i l u t i o n w i t h w a t e r , t h e r e was a parallel decrease in the optimum hernin concentration. When the lysate was diluted with fresh supernate, the hemin optimum remained the same. This indicated t h a t i t w a s t h e r a t i o of h e r o i n t o s o m e c o m p o n e n t i n t h e s u p e r n a t e , p r e s u m a b l y t h e proinhibitor, which was critical.
81
I
I
~f
I
I-
o
5
io
I
I
I
I
I
.|
+40~1 15
zo
H20
z5
HEMIN CONCENTRATION (I.LM)
-
3o
Fig. 8. Hemin optimum for globin synthesis in cell-free systems containing different proportions of lysate. The indicated proportions of lysate, supernate, and water were incubated with 3 °/~1 of master mix and 30/,I of water a t 34 °C for 90 min. The indicated hemin concentration was t h a t in the final incubation volume, I IO/[gl. Leucine incorporation represents hot trichloroacetic acidprecipitable radioactivity per I 1.4/zl of lysate. All incubations were performed in duplicate.
Biochim. Biophys. Acta, 287 (1972) 340-352
348
M. GROSS, M. RABINOVITZ
Rapid/ormation o/inhibitor induced by sullhydryl reagents Sulfhydryl reagents promoted a rapid formation and subsequent rapid inactivation of an inhibitory activity when incubated with the CMS-proinhibitor fraction and then neutralized with dithiothreitol (Fig. 9). When N-ethylmaleinfide or o-iodosobenzoate was mixed with the CMS-proinhibitor fraction during the course of the formation of the natural inhibitor, there was a rapid formation of additional inhibitory activity. This activity reached a maximal level of about 18oo units/ml at every time tested, the same level reached when the proinhibitor was completely converted to the inhibitor by warming alone (Fig. 9). Thus, as the amount of proinhibitor was depleted with its conversion to natural inhibitor, progressively less additional inhibitory activity could be induced by N-ethylmaleimide or o-iodosobenzoate. When CMS-proinhibitor had been fully converted to inhibitor by warming for 14 h at 34 °C, the addition of N-ethylmaleimide or o-iodosobenzoate produced no I00~
!
-
,
°o
6[)21
•
] 4[)
18
b
g
15
•
~ s
/ z
hibito¢
NEM-I nhlbdOr
.~I~.~-
o
~4~_~_~e.-~ ~ - ~
Standard Inhibitor 7 ~
~'o~'~ 6
%
~ 8 HOURS
I0
12
14
05
I'0 [15 [NHIB4TOR (/~1)
20
Fig. 9. Effect of sulfhydryl reagents on inhibitor formation. CMS-proinhibitor, prepared as indicated in Methods and at 3 times its c o n c e n t r a t i o n in the original supernate, was w a r m e d at 34 °C in the presence ( 0 - 0 ) and absence ( O - O ) of I 2 o / , M hemin. At the indicated times, 25-~1 aliquots of the sample i n c u b a t e d w i t h o u t h e m i n were mixed w i t h 25 ~ul of i.o • lO -3 M Nethylmaleimide ( a - / x ) or 2.5 • lO -3 M o-iodosobenzoate ([:]-[3), b o t h p r e p a r e d in IO times conc e n t r a t e d Buffer A. io-~ul aliquots of these m i x t u r e s were t h e n neutralized w i t h 5 #1 of o. 5 • lO -3 M or 2. 5 • lO -3 M dithiothreitol in IO times c o n c e n t r a t e d Buffer A, respectively, after 2, 6 and 15 rain. I n c u b a t i o n was c o n t i n u e d at 34 °C for 8 m o r e m i n u t e s before a s s a y i n g inhibitory a c t i v i t y w h i c h is expressed as u n i t s / m l of the original supernate. Neutralized N-ethylmaleimide, o-iodosobenzoate or dithiothreitol alone, at the levels used for e s t i m a t i o n of inhibitory activity had no effect on the a s s a y system. Fig. io. Dose dependence of inhibition p r o d u c e d b y o-iodosobenzoate-(OIB), N-ethylmaleimide(NEM), and s t a n d a r d inhibitor, o-Iodosobenzoate- a n d N - e t h y l m a l e i m i d e - i n h i b i t o r s were p r e p a r e d as described in Methods. S t a n d a r d i n h i b i t o r represents i n h i b i t o r y s u p e r n a t e which h a d been w a r m e d w i t h o u t h e m i n for 14 h at 37 °C and t h e n partially purified on CM-Sephadex in a m a n n e r identical to t h e p r e p a r a t i o n of p r o i n h i b i t o r (Methods). I t was t h e n lyophilized and dissolved in a v o l u m e of w a t e r e q u i v a l e n t to the v o l u m e of the original supernate.
Biochim. Biophys. Acid, 287 (I972) 340-352
INHIBITOR
OF
GLOBIN
CHAIN
349
INITIATION
more inhibitory activity. In addition, if CMS-proinhibitor was mixed with the sulfhydryl reagents and the reaction terminated with dithiothreitol within 15 min, further warming of this mixture produced no additional inhibitory activity. When CMSproinhibitor was incubated either with dithiothreitol or with the sulfhydryl reagents neutralized with dithiothreitol, the rate of formation of inhibitory activity was the same as that when CMS-proinhibitor was warmed alone, (data not shown). These results suggest that N-ethylmaleimide or o-iodosobenzoate, when added to CMSproinhibitor, rapidly convert the proinhibitor to a component which is similar to the natural inhibitor. When the dose of inhibitor, formed b y the action of N-ethylmaleimide or o-iodosobenzoate on CMS-proinhibitor, was varied, the degree of inhibition of cell-free hemoglobin synthesis followed the same semi-log relationship as that observed with 16 ~
]
~
--
I
T - - ]
I o.7
I
I
I
'°i
i
i 1
40 Vo I
1 1/
0.5
I
/
; ,~| ~ !?
(Hemoglobin
1
-
elutio, at 516ml)
I0 i30 ~
~'0.4
v B-
20 ~
0.3
i C~2
i 0.1
l
Ij~.%;:..~;" o
250
i
, "%.. ~2s
300 350 ELUTION VOLUME (ral)
400 MINUTES
F i g . i I. C h r o m a t o g r a p h y o n S e p h a d e x G - 2 o o of p r e c u r s o r for n a t u r a l i n h i b i t o r a n d t h a t i n d u c i b l e b y s u l f h y d r y l r e a g e n t s . A 145 c m x 2.5 c m S e p h a d e x G - 2 o o c o l u m n w a s p r e p a r e d a n d o p e r a t e d as i n d i c a t e d i n M e t h o d s . F r e s h s u p e r n a t e (4 m l ) w a s a p p l i e d , t h e c o l u m n d e v e l o p e d w i t h B u f f e r A, a n d 4 - m l f r a c t i o n s w e r e c o l l e c t e d . A f t e r d e t e r m i n a t i o n of t h e a b s o r b a n c e a t 28o n m , all f r a c t i o n s w e r e l y o p h i l i z e d a n d r e d i s s o l v e d i n 0.4o m l of w a t e r . I n h i b i t o r y a c t i v i t y w a s d e t e r m i n e d i n e a c h f r a c t i o n : w i t h o u t f u r t h e r t r e a t m e n t ( • - • • • ) ; a f t e r w a r m i n g a l i q u o t s a t 34 °C for 4 h ( O - - - O ) ; a n d a f t e r m i x i n g I O # 1 a l i q u o t s of e a c h f r a c t i o n w i t h IO p l of I . O . lO -2 M N - e t h y l m a l e i m i d e (zX- • - A ) o r 2. 5 • lO _2 M o - i o d o s o b e n z o a t e (VI- - -[~) i n IO t i m e s c o n c e n t r a t e d B u f f e r A, w a r m i n g for 2 m i n a t 34 °C a n d t h e n n e u t r a l i z i n g b y i n c u b a t i n g for a n a d d i t i o n a l 15 r a i n w i t h IO y1 of o. 5 • i o - s or 2.5 • lO -2 M d i t h i o t h r e i t o l , i n IO t i m e s c o n c e n t r a t e d B u f f e r A, r e s p e c t i v e l y . I n h i b i t o r y a c t i v i t y is e x p r e s s e d a s t h a t i n t h e o r i g i n a l , u n c o n c e n t r a t e d e l u a t e . F i g . 12. E f f e c t of h e r o i n o n i n h i b i t o r f o r m a t i o n b y s u l f h y d r y l r e a g e n t s . C M S - p r o i n h i b i t o r , i n o n e - t h i r d t h e v o l u m e of t h e o r i g i n a l s u p e r n a t e , w a s m i x e d w i t h a n e q u a l v o l u m e of i . o • I o -2 M N - e t h y l m a l e i m i d e a t o °C ( O , O ) , 2.5 • i o - I M o - i o d o s o b e n z o a t e a t 25 °C ( A , • ) , o r 2.0 • lO -2 M N - e t h y l m a l e i m i d e a t 34 °C (F1, It), a l l p r e p a r e d i n IO t i m e s c o n c e n t r a t e d B u f f e r A, i n t h e p r e s e n c e (closed s y m b o l s ) a n d a b s e n c e ( o p e n s y m b o l s ) of h e m i n , f i n a l c o n c e n t r a t i o n , 60 p M . A t t h e i n d i c a t e d t i m e s , io-~ul a l i q u o t s of e a c h r e a c t i o n m i x t u r e w e r e n e u t r a l i z e d w i t h 5/~1 of d i t h i o t h r e i t o l p r e p a r e d i n IO t i m e s c o n c e n t r a t e d B u f f e r A, a t c o n c e n t r a t i o n s of 0. 5 • lO -2 M, 2.5 • lO -2 M, a n d i . o • i o -~ M, r e s p e c t i v e l y , w i t h a d d i t i o n a l i n c u b a t i o n a t 34 °C for 15 m i n . I n h i b i t o r y a c t i v i t y was assayed in duplicate.
Biochim. Biophys. Acta, 287 ( I 9 7 2 ) 3 4 0 - 3 5 2
35 °
M. GROSS, M. RABINOVITZ
natural inhibitor (Fig. 1o). The component which becomes inhibitory when mixed with N-ethylmaleimide or o-iodosobenzoate co-chromatographs on Sephadex G-2oo with the natural proinhibitor (Fig. 1i). Heroin, however, only slightly retarded the formation of inhibitory activity when CMS-proinhibitor was incubated with either sulfhydryl reagent (Fig. 12). Several additional observations suggest that the inhibitory component, produced by the action of N-ethylmaleimide or o-iodosobenzoate on CMS-proinhibitor, m a y have the same mode of action as natural inhibitor. The inhibitory action of the former and that of the latter was prevented by a partially purified, 0.55 M KC1 wash of reticulocyte ribosomes 1~, I-fraction 14,2~ (Fig. 13). In addition, the inhibitor pro-
II I
I0 "
i
I
~0""~0~0 ~
I
I
I
O.
9I
g~
20 0 40 I-FACTOR {~g)
~o
60
Fig. 13. E f f e c t of p a r t i a l l y p u r i f i e d I - f a c t o r on t h e i n h i b i t i o n b y n a t u r a l , N - e t h y l m a l e i m i d e , a n d o - i o d o s o b e n z o a t e - i n h i b i t o r s . Cell-free s y s t e m s c o n t a i n e d t h e i n d i c a t e d a m o u n t s of I - f a c t o r a n d a p p r o x i m a t e l y I u n i t of n a t u r a l ( O - - - O ), N - e t h y l m a l e i m i d e - ([2]- - -[2] ), or o - i o d o s o b e n z o a t e ( A- - - A ) i n h i b i t o r in 3 ° / z l ; 3 °/~1 of m a s t e r m i x ; 5 ° / , 1 of l y s a t e a n d a fi na l he roi n c o n c e n t r a t i o n of 20/~M. C o n t r o l s a m p l e s ( 0 - - - 0 ) r e c e i v e d no a d d e d i n h i b i t o r . T h e p u r i f i e d I - f a c t o r 18 w a s a o.55 M KC1 e x t r a c t of r e t i c u l o c y t e ribosomes, d i l u t e d to o.I M KC1 w i t h buffe r (Tris-HC1, i o mM p H 7.5, d i t h i o t h r e i t o l , I raM; a n d o . i mM E D T A ) . The d i l u t e d e x t r a c t w a s c o n c e n t r a t e d b y u l t r a f i l t r a t i o n a n d a p p l i e d to a D E A E - c e l l u l o s e c o l u m n e q u i l i b r a t e d w i t h o. i M KC1 in t h e a b o v e buffer. A f t e r w a s h i n g t h e c o l u m n w i t h o.i M IZC1 in buffe r t o r e m o v e h e m o g l o b i n , i t w a s f u r t h e r w a s h e d , a n d t h e n t h e I - f a c t o r w a s e l u t e d w i t h o.16 M KC1 in buffer. I n c u b a t i o n s w e re c a r r i e d o u t a t 34 °C for 90 m i n a n d t h e h o t t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e r a d i o a c t i v i t y w a s d e t e r m i n e d in 25-/zl a l i q u o t s . N o t e t h e b r e a k in scale of t h e o r d i n a t e .
duced by N-ethylmaleimide action on CMS-proinhibitor caused complete disaggregation of polyribosomes within 5 min when incubated with lysate and an energy source at 34 °C, but had no effect in the absence of an energy source (Fig. 14). Natural inhibitor had an identical action on the polyribosomes. In neither case can the inhibitory activity be attributed to ribonuclease, which degrades the polyribosomes in the absence of an energy source. Biochim. Biophys. Acta, 287 (1972) 340-352
INHIBITOR OF GLOBIN CHAIN INITIATION
0.6[ A
°41/
351
F
c
i E
A2~oOB8 06I 02
CMFROMMENISCUS
Fig. 14. Effect of n a t u r a l a n d N - e t h y l m a l e i m i d e - i n d u c e d i n h i b i t o r s on t h e p o l y r i b o s o m e profile of r e t i c u l o c y t e lysates. Cell-free s y s t e m s , c o n t a i n i n g 15o/~1 of lysate, 9o/~1 of w a t e r (A, C, E) or m a s t e r m i x (B, D, F), a n d 15 i n h i b i t o r u n i t s of N - e t h y l m a l e i m i d e - i n h i b i t o r (C a n d D) or of n a t u r a l i n h i b i t o r (E a n d F) in 90/~1 of Buffer A, or 90/~1 of B u f f e r A alone (A a n d B), were i n c u b a t e d w i t h 2o/~M h e m i n a t 34 °C for 5 min. All s a m p l e s i m m e d i a t e l y received 300/~1 of 2-deoxyglucose, o . i o M, c o n t a i n i n g 15o u n i t s of h e x o k i n a s e 18, a n d were q u i c k l y cooled to o °C. 5oo~1 of each s a m p l e were l a y e r e d on a linear I5 to 3 ° % sucrose gradient. A f t e r c e n t r i f u g a t i o n a t 41 ooo rev./ m i n for lO 5 m i n in t h e S W 41 rotor, t h e c o n t e n t s of t h e t u b e s were p u m p e d b y u p w a r d d i s p l a c e m e n t t h r o u g h a flow cell in a B e c k m a n D U s p e c t r o p h o t o m e t e r , a n d t h e elution profile a t 26o n m w a s m o n i t o r e d w i t h a Gilford recorder.
Also tested was the possibility that other compounds which could influence the conformation of proteins would cause the rapid formation of inhibitory activity when added to CMS-proinhibitor. Treatment with 8 M urea, 5 M guanidine -HC1, or precipitation at p H 5 were all ineffective in promoting the rapid formation of inhibitory activity, (data not shown). These results suggest that some type of sulfhydryl reaction m a y be involved in the natural conversion of proinhibitor to inhibitor when supernate is warmed without heniin.
DISCUSSION
The complete conversion of proinhibitor to inhibitor, which normally requires incubation at 34 °C for 12 to 15 h, is rapidly accelerated b y such sulfhydryl reagents as N-ethylmaleimide and o-iodosobenzoate. By a number of criteria, the component upon which the sulfhydryl reagents act seems identical to the proinhibitor ,and the inhibitor whose formation they induce blocks globin chain initiation as does the natural inhibitor. The reactions occurring during the formation of natural inhibitor are still unknown, but our observations suggest that the change from proinhibitor to inhibitor m a y be the result of specitic conformational changes. First, the rate of the natural reaction is reduced b y less than a factor of 2 when the system which forms inhibitor is diluted b y a factor of 2o. Second, partial purification of the inhibitor fornfing activity b y ion exchange and gel filtration suggests that it m a y be one component, the proinhibitor. Therefore, we suggest that the reticulocyte contains some protein which, in the absence of hemin and at physiological temperatures, undergoes an alteration in conformation possibly involving disulfide bond formation 6r interchange, and becomes an inhibitor of globin chain initiation. In the course of this change, the inhibitory site on the proinhibitor m a y become unmasked. Biochim. Biophys. Acta, 287 (1972) 340-352
352
M. GROSS, M. RABINOVITZ
K o s o w e r et al. 2~ h a v e f o u n d t h a t o x i d i z e d g l u t a t h i o n e will i n h i b i t g l o b i n c h a i n i n i t i a t i o n i n a r a b b i t r e t i c u l o c y t e cell-free s y s t e m s u p p l e m e n t e d w i t h h e r o i n . W e t e s t e d w h e t h e r o x i d i z e d g l u t a t h i o n e w o u l d i n d u c e t h e f o r m a t i o n of i n h i b i t o r y a c t i v i t y w h e n i n c u b a t e d w i t h f r e s h s u p e r n a t e i n t h e p r e s e n c e of h e m i m T h e o x i d i z e d g l u t a t h i o n e w a s t h e n r e d u c e d w i t h d i t h i o t h r e i t o l . A c o n c e n t r a t i o n of g l u t a t h i o n e d i s u l f i d e , I 0 -a M, w h i c h i n h i b i t e d cell-free g l o b i n s y n t h e s i s m a x i m a l l y , g e n e r a t e d n o d e t e c t a b l e inhibitory activity in the supernate in 5 rain. Thus the inhibition by oxidized glutat h i o n e a p p e a r s t o b e m e d i a t e d b y a n o t h e r m e c h a n i s m . I t is of i n t e r e s t t h a t cell-free p r o t e i n s y n t h e s i s b y m i c r o s o m e s f r o m n o r m a l a n d r e g e n e r a t i n g l i v e r is also a f f e c t e d b y o x i d i z e d a n d r e d u c e d s u l f h y d r y l r e a g e n t s 26. T h e r e l a t i o n s h i p b e t w e e n t h e s e f i n d i n g s a n d t h o s e w i t h r e t i c u l o c y t e s will r e q u i r e f u r t h e r i n v e s t i g a t i o n .
REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 ':, 26
J. Kruh and H. Borsook, J. Biol. Chem., 220 (1956) 9o5. G. P. Bruns and I. M. London, Biochem. Biophys. Res. Commun., 18 (1965) 236. H. S. W a x m a n and M. Rabinovitz, Biochem. Biophys. Res. Commun., 19 (1965) 538. A. I. Grayzel, P. H6rchner and I. M. London, Proc. Natl. Acad. Sci. U.S., 55 (1966) 650. H. S. W a x m a n and M. Rabinovitz, Biochim. Biophys. Acta, 129 (1966) 369. H. S. Waxman, M. L. Freedman and M. Rabinovitz, Bioehim. Biophys. Acta, 145 (1967) 353. S. D. Adamson, E. Herbert and W. Godchaux, Arch. Bioehem. Biophys., 125 (1968) 671. W. V. Zucker and H. M. Schulman, Proc. Natl. Acad. Sci. U.S., 59 (1968) 582. S. D. Adamson, E. Herbert and S. F. Kemp, J. Mol. Biol., 42 (1969) 247. C. R. Maxwell and M. Rabinovitz, Biochem. Biophys. Res. Commun., 35 (1969) 79. M. Rabinovitz, M. L. Freedman, J. M. Fisher and C. R. Maxwell, Cold Spring Harbor Syrup. Quant. Biol., 34 (1969) 567. G. A. Howard, S. D. Adamson and E. Herbert, Biochim. Biophys. Acta, 213 (197 o) 237. C. R. Maxwell, C. S. Kamper and M. Rabinovitz, J. Mol. Biol., 58 (1971) 317 S. D. Adamson, P. M. Yau and E. Herbert, J. Mol. Biol., 63 (1972) 247. M. Gross and M. Rabinovitz, Proc. Natl. Acad. Sci. U.S., 69 (1972) 1565. M. O. Dayhoff and R. V. Eck, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Silver Spring, Md., 1967, 1968. C. R. Maxwell and C. Mencken, Anal. Biochem., 44 (1971) 235. S. Mizuno, J. M. Fisher and M. Rabinovitz, Bioehim. Biophys. Acta, 272 (1972) 638. W. V. Zucker and H. M. Schulman, Bioehim. Biophys. Aeta, 138 (1967) 400. A. S. Tavill, A. I. Grayzel, I. M. London, M. K. Williams and G. A. Vanderhoff, J. Biol. Chem., 243 (1968) 4987 . A. S. Tavill, G. A. Vanderhoff and I. M. London, J. Biol. Chem., 247 (1972) 326. H. M. Dintzis, Proc. Natl. Acad. Sci. U.S., 47 (1961) 247. M. Rabinovitz, and J. M. Fisher, Biochim. Biophys. Acta, 91 (1964) 313 . R. L. Miller and R. Schweet, Arch. Biochem. Biophys., 125 (1968) 632. N. S. Kosower, G. A. Vanderhoff, B. Benerofe, T. H u n t and E. M. Kosower, Biochem. Biophys. Res. Commun., 45 (1971 ) 816. R. D. Nolan and M. B. Hoagland, Biochim. Biophys. Acta, 247 (1971) 609.
Biochim. Biophys. Acta, 287 (1972) 34o-352