Effects of hemin and other porphyrins on protein synthesis in a reticulocyte lysate cell-free system

J. Nol. Biol. (1969) 42, 247-258

Effects of Hemin and Other Porphyrins on Protein Synthesis in a Reticulocyte Lysate Cell-free System ST~PNLEY D. ADAMSON, EDWARD HERBERT AND STEPIIEN F. KEMP University

of Oregon, Eugene, Oregou, U.S.A.

(Received 9 October 1968, and in revised form 27 January

1969)

In the absence of added hemin, the rate of protein synthesis in a lysate cell-free system from rabbit reticulocytes falls almost to 0 after 15 minutes incubation at 35°C and the proportion of ribosomes present as polyribosomes falls from 60 to about 15%. Addition of hemin at this time restores protein synthesis to 60 to 70% of the initial rate in the presence of added hemin, and polyribosomes to almost 80% of the initial level (40% ribosomes as polyribosomes). If hemin is added before the start of incubation, a high rate of protein synthesis is sustained well beyond 20 minutes and the conversion of polyribosomes to monoribosomes is greatly retarded or prevented. Metal derivatives of protoporphyrin IX have been synthesized and tested for ability to replace hemin in sustaining the rate of protein synthesis in the intact cell and in the lysate system. Aggregation of these compounds in the solution added to the lysate has been found greatly to influence their effectiveness in this respect. When care is taken to reduce aggregat’ion, it is found that the cobalt, nickel, magnesium and zinc derivatives will replace hemin in the lysate system, wherem the copper derivative will not. Fez+ and Co2+ will not replace hemin in this system. It has been shown previously that the latter ions do replace hemin in the intact cell. Cobalt protoporphyrin IX and protoporphyrin IX sustain protein synthesis in the intact cell as well as Fez+ or hemin. Magnesium and zinc protoporphyrin IX derivatives are about half as effective as Fez+ m sustaining protein synthesis, whereas the copper and nickel derivatives are not effective at all in this respect. Stimulation of protein synthesis by hemin is prevented by the addition of Pb” + to the lysate system. Concentrations of Pb 2f that prevent. the hemin effect also strongly promote aggregation of hemin.

1. Introduction The addition of ferrous ions to a suspension of rabbit reticulocytes increases the rate of synthesis of both heme and globin (Kruh & Borsook, 1956; Nizet, 1957; Morell, Savoie & London, 1958). This indicates that the two processes are linked to one another by the availability of Fez+ or a component with which Fez+ interacts. Cobalt can substitute for Fe2 + in maintaining the rate of globin synthesis but not in maintaining the rate of heme synthesis (Morel1 et al., 1958). 0th er results (Waxman & Rabinovitz, 1966) suggest that Fez+ affects globin synthesis after it is co-ordinated by protoporphyrin IX to form heme. In order to investigate these phenomena further, a highly active cell-free system has been developed similar to one described by Lamfrom 247

S. I>. ALIAMSON,

248

E.

HERBERT

AND

S. E’. KEMP

& Knopf (1964). The purpose of the present paper is to show t.llat’ tllis systcam cstri bit’s most of the regulatory effects of hemin on globin synthesis that, have been observed in the intact cell. It has been shown that reticulocyte lysate systems can synthcsizc protein at Iligh rates for 30 to 40 minutes if supplemented with hemin, and if phosphocrcatint: and creatine

kinase

are used to regenerate

ATP

(Adamson,

Godchaux

& Hcrbert8,

INS).

When such systems are incubated in the absence of added hcmin; the rate of protein synthesis declines sharply in the first 10 to 15 minutes and extSensivc polyribosomc breakdown occurs (Zucker & Schulman, 1968). The results in this paper show that hemin can restore globin synthesis and reverse polyribosomc hraakdown even aftctr the lysate system has been allowed to run down for 10 to 20 minutes. Hence, the dcca~ of protein synthesis does not involve extensive destruction of messenger RXA OI other labile component’s of the lysate. Thus, the stimulatory efYcct of homin iu the lysate system cannot be due merely to protection of a labile component from inactivation.

2. Materials and Methods (a) Preparution

of reticulocytcs

Reticulocytes were obtained from Eew Zealand white rabbits (female) made anemic by 7 daily injections of 2.5% (w/v) phonylhydrazine-HCI (Adamson et al., 1968). The amount of phenylhydrazine used was determined by the weight of the rabbit as follows: 5 to 5.5 lb, 0.8 ml./day; 5.5 to 6 lb, O.!) ml./day and above 6 lb, 1.01~1I./day. The animals were bled on t,he eighth day by heart puncture. The cells wcrc washed twice as previously described. (b) Preparation

of lysate

The method used to prepare the lysate has been described (Adamson el rzl., 19&S). Lysates from several rabbits were routinely pooled and stored in 5-ml. portions in liquid nitrogen. There was no appreciable difference in r.apacit,y to respond to hemin between the fresh and the frozen lysates. (c) hxbation The final incubation mixture consisted of 0.20 ml. of lysate; 0.050 ml. of a master mix solution, and 10 pl. of the component being tested (hemin, protoporphyrin IX, etc.) The master mix solution conbained the following components (final concentration of each component in the incubation mixture is given in parentheses): magnesium acetat,e (0.005 M) ; neutralized sodium salt of ATP (0.001 ~1); GTP (0.002 M); mercaptoethanol (0.0002 M) ; creatine phosphate (0.015 M) ; and creatine phosphokinase (60 enzyme units/ml.). ATP was obtained from the Calbiochem Corp., Los Angeles. GTP, creatine phosphate and creatine phosphokinase were obt,ained from the Mann Research Corp., Kew York. The fmal concentrations of non-radioactive amino acids in the master mix solllt,ion were one-tenth those used by Borsook, Fisher & Keighlcy (1957). The specific activity of L-[l%]leucine was between 5 and 15 pc/pmole in t,he final incubation mixtnrc. The leucine concentration was 1.5 x 10v4M. The amount’s of amino acids in the 1Ssatcs were considered to be negligible compared to the amounts added in the master mix solution. Uniformly labeled L-[14C]leucine was obt’ained from the Now England iYucloar Corp., Boston. (d) Preparation

of nkchllopor~hyrins

The protoporphyrin IX dimethyl esters of zinc, nickel and copper wore prepared by dissolving 0.25 m-mole of protoporphyrin IX dimethyl ester and 0.75 m-mole of met,al acetate in 20 ml. pyridine. This was sealed in an ampoule and incubat,ed at, 125°C for 12 hr. The ampoule was cooled and the contents filtered into 100 ml. of water, the prot,oporphyrin forming a precipitate. By this method the copper, zinc and nickel complexes could bc

PORPHYRIN

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249

prepared free of unreacted protoporphyrin IX dimethyl ester. However, in the case of magnesium protoporphyrin IX, Mg(ClO,)z was used instead of the acetate salt (Baum, Burnham & Plane, 1964), and further purification was required. In this case the precipitate obtained after the last step described above was centrifuged, dried and chromatographed as described by Wei, Corwin & Arellano (1962). Red-colored fractions were pooled and evaporated to dryness. The dry material was dissolved in 30 ml. ether and recrystallized as described by Falk (1964). The protoporphyrin IX dimethyl ester complex of cobalt was prepared in the manner described by Falk (1964). All metalloporphyrins had spectra in chloroform that corresponded closely to those already published, and furthermore, none of the spectra showed evidence of peaks at 505 and 630 rnp which would correspond to protoporphyrin IX dimethyl ester (Falk, 1964). Dimethyl esters of the metalloporphyrins were converted to the free acid forms as follows: 25 mg of the dimethyl ester were dissolved in 50 ml. of 1% KOH in methanol and 10 ml. of water were added (Falk, 1964). This mixture was refluxed for 5 hr, and the methanol was removed by evaporation. To remove remaining ester, the resulting basic solution was washed with chloroform until the chloroform layer was colorless. The aqueous layer was titrated to pH 4 with 3 N-HCl and the precipitate that formed was collected by centrifugation at 15,000 g for 10 min. The pellet was washed 3 times with distilled water and then dissolved in 0.1 ml. 1 N-NaOH, 0.1 ml. 0.5 M-Tris buffer (pH 7.8), and 2 ml. of 100% ethylene glycol. 75 ~1. of 1 N-HCl were Glen added slowly. The solution was centrifuged at 15,000 rev./min for 10 min and the supornatant liquid served as the metalloporphyrin added to the incubation mixtures. Protoporphyrin IX dimethyl ester was hydrolyzed by dissolving 100 mg of the ester in 50 ml. of 25qi; HCI and allowing this to stand at room temperature for 24 hr (Falk, 1964). Aggregation of the ester was brought about by titration to neutrality. The aggregates were large enough to be sedimented by centrifugation at 15,000 g. The sediment was resuspended in distilled water and centrifuged at 15,000 g for 10 min, and the supernatant liquid was used as the source of protoporphyrin IX added to the incubation mixtures.

I

!

,

(

150 475 500 525 550 575 600 Wavelength ! myd

I

625

FIG. 1. Effect of ethylene glycol on the spectrum of protoporphyrin IX. A. 6.3 X 10e4 M solution of protoporphyrin IX in water was diluted 1: 10 with 100% ethylene glycol (solid curve) or water (dashed curve). The spectra were recorded in a Cary model 11 spectrophotometer at room temperature.

260

(0) Ilelation

6. I).

ADAMSOX,

to state of aggreyatiolz

F:. HERBERT of protoporphyrins

AXL)

S. E’. KE&ll’

to ctrpacity

to sti~mulate protfi~L syktlrcsis

The porphyrin most difficult to keep in a d&aggregated state is protoporphyrin IX. We were not able to prepare the free acid form by basic hydrolysis without causing severe aggregation; in some preparations it was even possible t,o sediment the entire colloid bs centrifugation. Acid hydrolysis, which was routinely used, gave a less aggregated product. Ethylene glycol is helpful in keeping porphyrins in a disaggregated state. Figure 1 shows spectra of a preparation of protoporphyrin IX in aqueous solution and in ethylene glycol. Note the absence of sharp peaks in the spectrum in aqueous solution. This illust,rates the effect of ethylene glycol in helping to break up aggregates. Gel filtration chromatography also provides a general assay for state of aggregation. Highly aggregated preparations of protoporphyrin IX, such as the one which gives the spectrum in aqueous solution in Fig. 1, are retarded only slightly, if at all, on G75 Sephadex columns, while the preparations that appear to be disaggregated by the spectral criterion are difficult to remove from Gl5 Sephadex columns (&> 1). Secondary binding forces may be responsible for retarding the latter preparations. To ensure biochemical activity, it is important that the porphyrins be in a relatively disaggregated state. If an aqueous solution of hemin is allowed to stand at room temperature for several days, it becomes less effective in stimulating protein synthesis than fresh hemin preparations. Accompanying this loss of activity is the disappearance of a spectral peak at 490 rnp in water (Blauer & Rottenberg, 1963) with no change in the pyridine hemochrome spectrum (Falk, 1964). If a portion of the same hemin preparation is suspended in ethylene glycol (90% v/v) instead of water, the spectral peak at 490 m/l does not disappear on standing and the preparation does not lose t,he capacity to stimulate protein synthesis. Control experiments show that the small amounts of ethylene glycol added to the incubation mixture in the porphyrin solution do not affect protein synthesis. (f ) Reagents Hemin was obtained from the Eastman Co. Protoporphyrin IX dimethyl ester was obtained from the Mann Research Corp., New York. All metal:salts and organic reagents used were of reagent grade. Deuterohemin was a gift from Dr J. B. Neilands, of the University of California, Berkeley. (g) Counting

of radioactive

samples

To measure protein synthesis, lo-~1. or 20-~1. samples were removed from the incubation mixture and added to 8.0 ml. of 0.01 M-NaCl. This operation stopped the reaction. 20% trichloroacetic acid (containing 0.01 M non-radioactive L-leucine) was added to a final concentration of 5% and the samples were prepared for counting &9 described previously (Adamson et al., 1968). The measurement of “plateau levels” of protein synthesis in the experiments described in Fig. 4 and Table 2 was done as follows. Incorporation was determined at 60 and 70 min or 50 and 60 min after the start of incubation. Lack of significant differences in the incorporation at these times was taken as an indication that the rate of protein synthesis had fallen to essentially 0. The values were then averaged and the average value was taken as a measure of the plateau level of protein synthesis. Samples of fractions from the carboxymethyl cellulose columns were counted in a Nuclear Chicago scintillation spectrometer as previously described (Godchaux, Adamson & Herbert, 1967).

3. Results (a) Reactivation of protein synthesis and reaggregation of polyribosomes by addition of hemin Experiments were performed in which hemin was added to lysates in complete master mix solutions at various times after the start of incubation at 35°C. Separate samples were withdrawn for determination of the rate of protein synthesis and

PORPHYRIN

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251

polyribosome content. Although the absolute amount of stimulation of protein synthesis by hemin varied from one lysate to another as described more fully below, they all showed the following behavior. First, protein synthesis was stimulated to a significant extent by hemin; and second, the earlier the hemin was added the greater the stimulation of protein synthesis observed. Figure 2 illustrates the effect of hemin on the most responsive lysate studied. In this case the rate of protein synthesis fell almost to 0

1 11. 7,'20

60

16 P 0 12 2 F Y ” 8

4

Time (mm) FIG. 2. Effect of late addition of hemin on protein synthesis. Hemin was added to lysate incubation mixtures which had been incubated at 35’C in the absence of hemin for 0 min (-e-e-), 5 min (-i--n-), 10 min (-.+-A-), 15 min (-@J-&-) -) and 20 rnin (-O-O-), to give a final concentration of 5 x 10M5 11. One mixture (-x-x was incubated for the entire period in the absence of hemin. Samples were removed at t,imes indicated and protein synthesis was determined as described in the Materials and Methods section. The specific activity of [14C]leucine used was 6.7 ~c/~mole.

after about 12 minutes incubation at 35°C in the absence of added hemin. Addition of hemin at 20 minutes restored the rate of protein synthesis to about 70% that of the sample to which hemin had been added at 0 time. Figure 3 shows the effect of hemin on the polyribosome content of samples removed from the no hemin and the 15- and 20-minute incubation mixtures referred to in Figure 2. Note in Figure 3 that ra,pid disaggregation of polyribosomes coincides with a rapid decline in the rate of protein synthesis (Fig. 2). After 15 minutes incubation in the absence of hemin, the polyribosome content was 25 to 30 “/o that of the sample incubated in the presence of added hemin. The addition of hemin after 20 minutes restored the polyribosome level to greater than 70% that of the mixture containing hemin from the start. Addition of hemin at the beginning of incubation caused a slight rise in polysome content, followed by a fall after 30 minutes. The stimulation of the plateau level of protein synthesis observed in the past several months with more than twenty lysates was greater than l-5-fold when

252

S. D.

ADAMSON,

E. HERBERT

AND

No additions

0.10 I

S. E’. KEME

5

IO

I 15 20

I 25

30

35

I

40

Time (min)

FIG. 3. Effect of late addition of hemin on polyribosomes. In the experiment described in Fig. 2, 0.2-ml. samples were also removed from the incubation mixtures to which hemin had been added at 0 min (-n-n-), 15 min (- x - x -), and 20 min (-O-e--). 0.2-ml. samples were also removed from the mixture which had received no hemin (--O-O-). These samples were added to 0.5 ml. of standard ribosomal buffer (Adamson et al., 1968) and 0.3 ml. of this mixture was layered on a 15 to 30% sucrose gradient and centrifuged for 45 min at 50,000 rev./min in an SW50 rotor of a Spinco model L2 HV ultracentrifuge. The Azso of fractions collected from the gradient was determined as described previously (Godchaux et al., 1967).

hemin was added at the beginning of the incubation period. Of these lysates, more than half showed stimulation by hemin by more than 2*5-fold. Several of the latter lysates were studied further by the technique shown in Figure 2, except that fewer time points were analyzed. Most of these lysates showed a response similar in magnitude to that in Figure 2 when hemin was added within five minutes after the start of incubation, and somewhat smaller responses when hemin was added 10, 15 or 20 minutes after the start of incubation. These responses are relative to the response of the lysate at 0 time. Only one of these lysates showed no response after five minutes incubation. The results obtained with any one lysate are highly reproducible, in contrast to variability between lysates. Considerable variation from lysate t’o lysate is also observed in the effect of hemin on polyribosome content, but the trends seen in Figure 3 are always noted. Determination of radioactivity in polyribosome fractions from density gradients shows that the polyribosomes that re-form after addition of hemin carry nascent peptide chains (Adamson et al., 1968). Calculations of the synthetic time (time required for a functional unit or active ribosome to synthesize one complete peptide chain) as described previously (Adamson et al., 1968), utilizing measurements of poIyribosome specific activity as well as the data shown in Figures 2 and 3, indicate that when hemin is added at 15 or 20 minutes, the rate of protein synthesis per functional ribosome approaches one minute per chain -a value typical of samples to which hemin is added at time 0. Thus, the slightly lower rate of protein synthesis observed when the hemin is added at later times (Fig. 2) is due entirely to a smaller number of functional ribosomes. Also note that the rate of protein synthesis proceeds almost linearly for more than 10 minutes after

PORPHYRIN

EFFECTS

IN

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CELL-FREE

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2.3

addition of hemin at 15 or 20 minutes. This suggests that each functional unit synthesizes at least 10 globin chains after protein synthesis is restored. Hence, hemin restores the capacity of the system to make new chains and not just the capacity to complete chains already started on the ribosomes. Carboxymethylcellulose chromatography (Godchaux et al., 1967) of a lysate sample which had been incubated for 20 minutes in the absence of added hemin and radioactive amino acid, and then in their presence for an additional 40 minutes, shows that at least 75% of the protein synthesized during the recovery period can by accounted for as a- and p-chains of hemoglobin. More detailed studies of the nature of the protein products formed in the lysate syst’em have been reported (Zucker & Schulm.an, 1968; Adamson et al., 1968). TABLE Effects qf protopwphyrin

IX

Final concent rat,ion

Addition

Molarity 2x10-4

Fe2 + NOW2 Hemin Hemin Hcmin Protoporphyrin Protoporphyrin Protoporphyrin Protoporphyrin Mg Protoporphyrin &Ig Protoporphyrin Jig Protoporphyrin

IX IX IX IX IX IX IX

&Iolarity 5 x 10--s 1 x 10.-4 2 x 10 -- 4 Molarity 1.3x IO-4 2.5x 10-4 3.8x 1O-4 5.0x IO-4 AIolarity 1.8 x 10-S

3.5 Y IO-5 7.0x IO-5

A399 IX IX IX

11 22 33

(‘0 Protoporphyrin ( ‘o Protoporphyrin

IX IX

A 114 11 t’h A 385

(‘II

IX

Zn Zn Zn

Protoporphyrin Protoporphyrin Protoporphyrin

Protoporphyrin

1

compounds on protein

IX

synthesis in the intact cell Rrlatirc protein synthrris

1.00 by definition 040

O.i8 1.10 1.14 0.21 0.68 1.14 O,!)d

0.06 0.17 0.16

0.33 (I.34 041

0.49 0.99 0.01

A 343

Ki

Protoporphyrin

IX

14

0.01

In these experiments intact reticulocytes wrc~re incubated in the presence of L-[‘4C]leucinc (1 pc/pmole) and various protoporphyrin compounds (Godchaux et al., 1967). In each experiment, two control samples were incubated without added protoporphyrin IX compounds. One control metals. Protein samples sample cont,ained 2 x 10m4 M-Fe’+ ; the other had no added transition were taken at 15-min intervals, though only tho 60-min points were used in the preparation of this Table. Counting was done as described in the Materials and Methods section. For ease (;f presentation, the value for the 2 x 10y4 &I-Fe 2+ incubation is defined as 1.00 in each experiment, and the value for incorporation in the absence of added transition m&al is defined as 0. The values for incorporation in the presence of added protoporphyrin IX compounds were then converted to tQis Iscale within each experiment. The average amount of radioactivity at 60 min was about 4 x lo5 cts/min/ml. cells when Fe2 + was present and 2.5 x lo5 ctslminiml. cells when no additions were made.

254

S. D.

ADAMSON,

E.

HERBERT TABLE

AND

None Hemin Zn protoporphyrin Co protoporphyrin Mg protoporphyrin Ni protoporphyrin Cu protoporphyrin Protoporphyrin IX Deuterohemin

IX IX IX IX IX

KEMP

2

Effect of metalloporphyrins

Addition

S. F.

on protein synthesis

Concentration that produced maximum stimulation

Cta/min per ml. lysate x 10-4

-

1.1 5.6 5.9 4.6 4.4 4.9 1.7 4.8 4.8

4X10-sM A 399 =lfv A 411=9.5t 3.5 x 10-S M Am = 19t A m=w 1.5 x 10-4 M 3,2x 1O-5 JI

The concentration of each metalloporphyrin listed in the Table is the one that gives a maximum level of incorporation of leucine when tested over a broad concentration range. The incubation procedure and determination of the plateau level of incorporation were carried out as described in the Materials and Methods section. The specific activity of [‘%]leucine used was 6.7 pc/pmole. 7 Because extinction coefficients for the Zn, Co, Ni and Cu complexes of the free acid form of protoporphyrin IX were not available, their concentrations are reported as absorbancy of the Soret peak in aqueous solution. Small changes in ionic strength or pH will shift this peak. However, when solutions prepared as described in the Materials and Methods section are diluted with water, the positions of the peaks are reproducible within 2 rnp.

(b) Eflect of various metalloporphyrins

on protein synthesis

Protoporphyrin IX derivatives of zinc, magnesium, cobalt, copper and nickel were prepared as described in the Ma.terials and Methods section. The stimulatory effect of each of these complexes as well as hemin and deuterohemin was determined in the intact cell (Table 1) and in the lysate system (Table 2) over a broad concentration range and under conditions described in the Materials and Methods section to minimize aggregation. Waxman & Rabinovitz (1966) previously found that zinc protoporphyrin IX and deuterohemin were as effective as hemin in supporting globin synthesis in intact cells, whereas copper protoporphyrin IX, copper, manganese and palladium deuteroporphyrins and protoporphyrin IX were without effect. Nickel deuteroporphyrin was partially effective. The most significant extension of the above work in this study is the finding that cobalt protoporphyrin IX is fully active in supporting protein synthesis in the intact cell (Table 1). The only major difference from the above mentioned findings is the observation that, if care is taken to avoid the formation of high molecular weight aggregates, protoporphyrin IX itself is as active as hemin in supporting protein synthesis. Magnesium protoporphyrin IX consistently shows a small stimulation of protein synthesis, whereas no stimulation is observed in the case of either the copper or the nickel derivative of protoporphyrin IX. Some caution should be exercised in the interpretation of negative results in view of the importance, again, of aggregation. The concentration of metalloporphyrin that produces maximum stimulation when added to the lysate at time 0 is given in Table 2. Note that the concentration of protoporphyrin IX causing maximum stimulation is four times that of the hemin concentration producing maximum stimulation. Since hemin is a likely contaminant

PORPHYRIN

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of protophorphyrin IX preparations, one might argue that hemin contamination is causing the stimulation observed. However, 3 x 10 -5 M-protoporphyrin IX produces slightly greater stimulation than 6 x 10e6 &I-hemin and hemin stimulation falls off sharply below the latter concentration (unpublished results). Protoporphyrin IX preparations then would have to contain roughly 20% as much hemin as protoporphyrin IX for this explanation to be correct. Determination of the Fez+ content of protoporphyrin IX preparations (Snell & Snell, 1949) shows that less than 5 m-moles of hemin can be present per mole of protoporphyrin IX. This excludes the possibility that added hemin causes the stimulation. However, small contamination with other porphyrin compounds, such as mesoporphyrin, cannot be excluded at this point. In other experiments, Fe2 + and Co2+ were also tested for stimulation of protein synthesis. In most lysates Fe2 + caused about 1*2-fold stimulation. The highest stimulation observed was 2-fold (2 x 10m3 M-Fe2+). Concentrations of Co2+ from 1 x 10e6 to 2 x 10 -’ M failed to stimulate protein synthesis in the cell-free system. Thus, the results in the lysate system fall essentially into two categories: the zinc, magnesium, iron, cobalt and nickel complexes of protoporphyrin IX, protoporphyrin 1.X itself, and deuterohemin, all give approximately the same level of stimulation of protein synthesis. Copper protoporphyrin IX, Co2+ and Fez+ generally give little or no stimulation, although in the case of Fe2 + significant stimulation is observed in some lysates. (c) Effect of lead ions on protein

synthesis

Previous studies by Waxman & Rabinovitz (1966) have shown that lead ions inhibit protein synthesis and cause breakdown of polyribosomes in intact reticulocytes. Figure 4 illustrates the effect of 1 x 10 -4 M-Pb2 + on protein synthesis in lysate

I -x

lO-4 M-Pb”

-X --”-. 1. -*-.v.

lo-3 r-pj,2+

I 5 x 10-S Concentration

I 10-4 I.sxlo-4 of hemin (molar)

i < ~I’ ’ Oz I

J

FIG. 4. Effect of Pba+ on protein synthesis. Hemin was added to 3 series of incubation mixtures to give the concentrations shown. One series contained no Pba+ (-O--O-), another contained 1 x 10e3 al-Pb2+ (-@--a-), and a -). Plateau levels of protein synthesis were determined as third lx 1O-4 r.r.Pba+ (-X-X described in the Materials and Methods section. Specific activity of [14C]leucine was 6.67 &pmole. The Pba + acetate salt was used.

S. I).

“56

ADAMSOX,

E. HERBERT

AND

S. F. KEI1If’

incubation mixtures containing different concentrations of hemin. The no additions curve shows the stimulatory effect of the same concentrations of hemin in the absence of added Pb2 + . Each point, represents a plateau level of [14CJlcucine incorporation as described in the Materials and Methods section. The levels of prot,ein synt.hesis in these curves are nearly the same as the level in the absence of added hemin. suggesting that Pb ‘+ interferes with the stimulation of protein synthesis by hemin. (d) Effect of lead ions m aggregation

of hensi~

The interactions of lead ions and hemin were observed spectrally by adding various concentrations of Pb2+ to solutions of hemin (5 x 10 -5 M) in 10% ethylene glycolconditions which favor disaggregation. The results are given in Figure 5. With solutions of hemin, the small peak at 490 rnp is diagnostic for a disaggregated preparation as described in the Materials and Methods section. As little as 6 x 10 -5 M-Pb2 +

I

1 450

475

500

I 525

550 575 Wavelength (m,d

600

I 625

t ;0

FIG. 5. Effect of Pb2+ on the spectrum of hemin. Pb2+ was added to 5 x 10m5 M solution of hemin to give concentrations of 1 x 10m5M (------1 and 5 x IO-5 M (- - - -). The dotted cm-ve (. . . . . . .) represents a solution of hemin with no added Pb2 + . The spectra were immediately determined as described in Fig. 1. The Pb2 + acetate salt was used.

was sufficient to convert this peak to a shoulder, and 1 x 10 -4 M-Pb2+ obliterated of the this peak. Slightly higher concentrations of Pb2 + caused visible precipitation hemin. Furthermore, iwhen hemin in 5 x 1O-5 M-Pb2 + is added to a G75 Sephadex column, it comes off the column in the void volume (K, N 0).

4. Discussion The observation that, after a period of incubation in the absence of hemin, some lysates are still responsive to addition of hemin makes several points. Hemin is clearly not merely protecting some labile component of the system from irreversible inactivation, for if this were so, no stimulation would be noted after the incubation period in the absence of hemin. For example, reaggregation of polyribosomes after initial breakdown and stimulation of hemoglobin synthesis show that the messenger RNA is still intact, and hence hemin is not merely inactivating nucleases. Note that

PORPHYRIN

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SYSTEM

25;

less than 0.5 mpmole of leucine is incorporated per ml. of lysate between 20 and 40 minut,es in the absence of hemin (no-hemin sample in Fig. 2), whereas 22 mpmoles is incorporated in the presence of hemin (20-minute sample in Fig. 2) during this same time interval. This high sensitivity to hemin after a 20-minute incubation period argues for an important role for hemin in the synthesis of new globin chains. For reasons previously discussed, the site of hemin action is likely to be at the level of attachment of ribosomes to polyribosomes, chain initiation, or a related process. Little is known yet about the details of the hemin requirement. The react’ions involved are very sensitive to the chemical environment. Previous reports (Zucker & Schulman, 1968; Adamson et al., 1968) that the hemin response of a number of lysatcs was lost faster than protein synthetic capacity, turn out, in our hands, to br dependent, on the LEX of t*hc phosphocnolpyruvate-ATP regeneration system? for these results have been confirmed with the same lysatc, which, when used with a creatine phosphnt,t system, generates Figures 1 and 2. The reasons for the &riking differcncc betnccsn the two energy syst’ems are unknown. To assess whet’hcr formation of hemin is required for stimulation of protein synthesis in in-tact reticulocytes, attempts have previously been made t,o inhibit insertion of Fe2 + int,o protoporphyrin IX by use of Pb 2 + . The finding that the sa,mc concentrations of Pb”+ that inhibit the hemin effect in the lysate cell-free system strongly promote t,hc aggregation of hemin suggests that a great deal of caution should be exercised in drawing conclusions about the inhibitory act’ion of Pb 2 + in t,he above system. Under the ionic conditions that exist in the cell-free system or t,he intact cell, there is littlc likelihood that much hemin would exist as monomer. It should be apparent, that in testing any new porphyrin compound it is very important to establish criteria for is instate of aggregat,ion, particularly before concludin g that a given porphyrin effect.ive in stimulating protein synthesis. Protoporphyrin IX, it,self, is particularly difficult ,to obtain in disaggregated form, and not surprisingly, therefore, is usually found to be inact#ive, both in our hands (Aclamson et al., 1968) and in t’he hands of other investigators (Waxman & Rabinovitz, 1966 ; Zucker & Schulman, 1968). It is not known whether the ability of the disaggregated preparations of protoporphyrin IX to stimulate protein synthesis involves its conversion to hemin first, but a fivefold excess of bipyridinc fa’iled to block the effect of protoporphyrin IX on t’he lysatc cell-f&e syshcm. The only porphyrin examined which gives no evidence of aggregation and still fails to stimulate prot,ein synthesis extensively in the lysate system is copper protoporphyrin IS. Attempts to correlate activity in the cell-free system with known chemical properties of the porphyrins such as ligand binding have not, as yet, been successful. Final],\-, the observation that Co 2+ fails to stimulate the cell-free system at all, and Fe2 + produces only a small stimulation, is further evidence that t’he rffect)s of these metal ions in the intact reticulocyte involve incorporat,ion into more complts molecules and arc not actions of the free ions. $ metalloporphyrin is an obvious candida& for the active molecule. Of the transition metals t’csted (Zn2 + , Cr” +, Ni2 +, &I'+, Mn2+, Co”+, Fc2+), only Fe2+ or Co2+ is effective in &mulating protein spnthesi8s in intact reticulocytes, whereas all metalloporphyrins tested except copper protoporphyrin IX stimulate the cell-free system. Enzymes from liver mitochondria have been observed bo catalyze the insertion of Fe2 + and Co2 + into protoporphyrin. but not, Mg2 + , Ca2 + , Ni2 +, Cd2 + , Pb2 + , Cu” + , &In2 +, Zn2 + or Hq2 + (Labbe & Hubbard. 1961). Thus, if t,he active molecule were a metalloporphyrin, an enzyme such as

258

8. D.

ADAMSON,

E.

HERBERT

AND

S. F.

KEMI‘

the liver enzyme would be able to make use of Co2+ or Fe2+ in the intact cell hut of no other metal ion. However, chemically synthesizecl metalloporphyrins of many types might be expected to show activity. If the enzyme system that inserts the metals into protoporphyrin IX is associated with mitochondria in reticulocytes, then it would be removed during preparation of the lysate which involves a sedimentation step (20,000 g). This would then explain why Fe2 + and Co2+ do not stimulate protein synthesis in the cell-free system. All of the porphyrin compounds that stimulate protein synthesis effectively in the intact cell (Table 1) stimulate protein synthesis effectively in the lysate system (Table 2). However, Zn2 + and Mg2 + protoporphyrins are much more effective in the lysate system than in the intact cell. We cannot suggest a definitive explanation f’or the latter finding at this point. We thank Judy Holt and Peter Yau MO-Ping for their excellent technical assistance. Thanks are also due to Allan Larrabee for critical advice in the preparation of the manuscript. This investigation was supported by U.S. Public Health Service research grant CA-07373 from the National Cancer Institute, and National Science Foundation grant GB-4063. One of us (E. H.) is the recipient of a Public Health Service Research Career Program award (l-KG-CA-2101), and one of us (S.D.A.) is a Public Health Service Predoctoral Trainee. REFERENCES Adamson, S. D., Godchaux, W., III & Herbert, E. (1968). Arch. Biochem. Biophys. 125, 671. Baum, S. J., Burnham, B. F. & Plane, R. A. (1964). Proc. Nut. Acad. Sci., Wush. 52, 1439. Blauer, G. & Rottenberg, H. (1963). AC&. Chem. Stand. 1’7, S216. Borsook, H., Fisher, E. H. & Keighley, G. (1957). J. Biol. Chem. 229, 1059. Falk, J. E. (1964). Porphyrins and Metdloporphyrins. Amsterdam, London & New York: Elsevier Publishing Co. Godchaux, W., III, Adamson, S. D. & Herbert, E. (1967). J. Mol. BioZ. 27, 57. Hruh, J. & Borsook, H. (1956). J. BioZ. Ohem. 220, 906. Labbe, R. F. & Hubbard, N. (1961). Biochim. biophys. Actu, 52, 130. Lamfrom, H. & Knopf, P. (1964). J. Mol. BioZ. 9, 558. Morell, H., Savoie, J. C. & London, I. M. (1958). J. BioZ. Chem. 233, 923. Nizet, A. (1957). Bull. Sot. Chim. BioZ. 39, 265. Snell, F. D. & Snell, C. T. (1949). In Calorimetric Methods of Analysis, vol. 2, p. 300. New York: D. Van Nostrand, Inc. Waxman, H. S. & Rabinovitz, M. (1966). Biochim. biophys. Acta, 129, 369. Wei, P. E., Corwin, A. H. & Arellano, R. (1962). J. Organic Chem. 27, 3344. Zucker, W. V. & Schulman, H. M. (1968). Proc. Nat. Acad. Sci., Wash. 59, 585.