Proc. Nat. Acad. Sci. USA Vol. 72, No. 12, pp. 4849-4853, December 1975

Biochemistry

Association of a cyclic AMP-dependent protein kinase with a purified translational inhibitor isolated from hemin-deficient rabbit reticulocyte lysates (protein synthesis control/regulation by hemin/phosphorylation of histone)

DANIEL H. LEVIN, RAJINDER SINGH RANU, VIVIAN ERNST, MICHAEL A. FIFER, AND IRVING M. LONDON Department of Biology, Massachusetts Institute of Technology, and Department of Medicine, Harvard Medical School, and Harvard-M.I.T. Program in Health Sciences and Technology, 77 Massachusetts Avenue, Cambridge, Mass. 02139

Contributed by Irving M. London, September 22, 1975

In the absence of added hemin, protein synABSTRACT thesis in rabbit reticulocyte lysates proceeds at maximal linear rates for several minutes and then ceases abruptly. Inhibition involves the action of a translational inhibitor whose formation is regulated by hemin. Addition of the isolated inhibitor to hemin-supplemented lysates produces an inhibition of protein chain initiation similar to that observed in heme-deficiency. The inhibitor has been purified over 300fold and contains a protein kinase activity that copurifies with the inhibitory function. With calf thymus histone II as the phosphate receptor, the inhibitor-associated protein kinase requires ATP as the phosphorylating agent. Cyclic AMP stimulates kinase activity 5- to 8-fold; the concentration of cyclic AMP required for half-maximal activity is 4 'X 10-8 M. Preincubation of the inhibitor in the presence of cyclic AMP significantly reduces cyclic AMP-dependent phosphorylation and inhibitory activity. The corresponding protein' kinase activity from hemin-supplemented lysates displays reduced cyclic AMP-dependency and little or no inhibitory'activity. These findings suggest that the protein kinase activity associated with the purified translational inhibitor is involved in the mechanism of inhibition of initiation observed in hemedeficient reticulocyte lysates.

Previous studies have established that the inhibition of protein synthesis observed in heme-deficient rabbit reticulocyte lysates (1-5) is reversible by hemin, but with decreasing efficiency as hemin addition is delayed (4-6). The inhibition is due in part to the action of a translational inhibitor (2, 4, 7-9) which can be induced in postribosomal extracts (S150) of heme-depleted lysates (2, 3, 7-10). The inhibitor formed after prolonged induction has been purified over 300-fold (11) (R. S. Ranu and I. M. London, manuscript in preparation). Addition of the purified inhibitor to hemin-supplemented lysates produces inhibition kinetics similar to hose induced by heme-deficiency (1, 3, 4), double-stranded RNA (dsRNA) (12-16), and oxidized glutathione (GSSG) (15, 17, 18). All four types of inhibition appear to be exerted through similar mechanisms (4, 9, 15, 19-21); the evidence suggests that the site of inhibition involves an early step in protein chain initiation (19-21). The recent findings that cyclic AMP (cAMP) also reverses the inhibitions induced by hemedeficiency, dsRNA, GSSG (22), and the purified inhibitor (V. Ernst and I. M. London, manuscript in preparation) further support the hypothesis of similar inhibitory mechanisms. In this report, we present evidence that a cAMP-dependent protein kinase (PK) activity is associated with the translational inhibitor isolated from heme-deficient lysates. Several observations suggest (a) that the cAMP-dependency Abbreviations: PK, protein kinase; cAMP, cyclic 3':5'-AMP; cGMP, cyclic 3':5'-GMP; dsRNA, double-stranded RNA; GSSG, oxidized glutathione; buffer A, 20 mM Tris-HCI (pH 7.6)-50 mM KCI-10%

glycerol- 1 mM dithiothreitol.

4849

of the protein kinase activity is linked to the inhibitory function, and (b) that hemin in some manner affects the functions of one or more protein kinase activities. A model to define these relationships is proposed and discussed. MATERIALS AND METHODS Preparation of Reticulocyte Lysates. Reticulocytes were obtained from rabbits treated with 1-acetyl-2-phenylhydrazine (Sigma Chemical Co., St. Louis, Mo.), and lysates were prepared as described (4). AMP, GMP, ATP, GTP, and calf thymus histone II were obtained from Sigma Chemical Co. [14C]leucine, ['y-32P]ATP, and ['y-32P]GTP were purchased from New England Nuclear (Boston, Mass.); and hemin from Calbiochem (Los Angeles, Calif.). Protein Synthesis In Vitro in Reticulocyte Lysates. Protein synthesis was assayed in 50-i1 reaction mixtures at 300 by the uptake of [14C]leucine (182 mCi/mmol; 312 cpm/ pmol) into lysate protein (4, 21). Assays contained 20 ,uM hemin unless otherwise stated. Assay of Translational Inhibitor. A translational inhibitor was purified over 300-fold from heme-deficient lysates (11). Details of the purification procedure will be described elsewhere (R. Ranu and I. M. London, manuscript in preparation). Each inhibitor fraction was titrated in the standard incubation in the presence of 20 ,M hemin to determine the gg of inhibitor protein that reduced ['4C]leucine incorporation to the level of controls without hemin- (see Fig. 1 and Table 1). Purification of Two Protein Kinase Activities from Hemin-Supplemented and Heme-Deficient Reticulocyte Lysates. A partial purification of two protein kinase activities from both hemin-supplemented and heme-deficient reticulocyte lysates was carried out at 40 by a modification of methods previously described (23-26). Each step of the purification procedure was the same for both lysates except that one lysate was fractionated in the presence of 10 ,uM hemin. Two 1-ml portions of a freshly thawed lysate were centrifuged at 150,000 X g for 3 hr to obtain the S150 extracts. The heme-deficient S150 fraction was incubated at 350 for 2 hr to induce formation of the translational inhibitor. Each S150 extract was fractioned with (NH42S04 to obtain the 0-50% saturated fractions, which were pelleted, dissolved in 1 ml of 20 mM Tris-HCI (pH 7.6)-50 mM KC1-10% glycerol-i mM dithiothreitol (buffer A), and dialyzed separately against buffer A. Each fraction was loaded on separate phosphocellulose (BIO-RAD, Richmond, Calif.) columns (0.9 cm X 6 cm) equilibrated with buffer A. The initial effluent protein fraction which contained the protein kinases (26) was loaded directly onto separate DEAE-cellulose (BIO-RAD)

4850

Biochemistry:

Levin et al.

Proc. Nat. Acad. Sci. USA 72 (1975)

+hemin

Table 1. Copurification of a protein kinase activity with reticulocyte translational inhibitor

O 750_ 0

a:/ 0~ *

o 0

Z 500_

Purification step

z/ ;

LL250

.O L

/

+0.84,og

fradion

-6/§

10 MINUTES

FIG. 1. Effects of a purified reticulocyte translational inhibion the kinetics of protein synthesis in a hemin-supplemented reticulocyte lysate. Reaction mixtures (50 Mul) for protein synthesis were prepared as described in Materials and Methods. Aliquots (5 ,Ml) were removed at intervals and assayed for [14C]leucine incorporation into lysate protein; values in the figure are adjusted for pmol incorporated per 50 Ml. *, Control + 20 MM hemin; 0, minus hemin; X, + 20 MAM hemin and 0.84 ,ug of Fraction 3 inhibitor (see Table 1). tor

columns (0.9 X 8 cm) equilibrated in buffer A, followed by elution in a stepwise gradient in buffer A with increasing concentrations of KC1. In both fractionations two protein kinase fractions were separately eluted at 0.1 M KC1 (PK-I) and 0.24 M KCl (PK-II) (see Table 4). PK-II of the hemedeficient lysate contained the translational inhibitor. Assay for PK In Vitro. The assay for PK activity is based on the covalent incorporation of the gamma phosphate of [,y-32P]ATP into histone II and is a modification of assays previously described for reticulocyte (23-26) and beef heart muscle (27) kinase activities. Assays contained the following components in a final volume of 50 ,l: 25 mM K+-Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate) (pH 7.15) buffer, 6 mM magnesium acetate, 30 ,ug of crystalline bovine serum albumin (Pentex Co.), 50 Mg of histone II, and 0.4 mM [y-32P]ATP or [y-32P]GTP with specific activities adjusted to an average of 75 mCi/mmol; cpm/pmol was determined for each experiment by absorbing aliquots of the reaction mixtures on glass fiber filters (GF/A, 24 mm, Whatman Ltd.), which were dried and counted in Liquiflor (New England Nuclear) in a liquid scintillation spectrometer (Searle/Nuclear Chicago). The reaction was started by the addition of an amount of enzyme that gave linear kinetics Of 32p incorporation for at least 15 min at 300. Reactions were stopped at 15 min by the addition of 1 ml of cold 7.5% trichloroacetic acid containing 1 mM NaH2PO4, left at 00 for 5 min, and then heated at 90° for 5 min. After cooling in ice, the suspensions were collected on glass fiber filters, washed with cold 5% trichloroacetic acid containing 1 mM NaH2PO4, and dried. Radioactivity was determined as above. Except where noted, results were expressed as pmol of 32P incorporated into histone per Ag of enzyme. RESULTS Effect of a Purified Reticulocyte Translational Inhibion Kinetics of Protein Synthesis in a Hemin-Supplemented Reticulocyte Lysate. Fig. 1 demonstrates the effect of 0.84 Mg of inhibitor Fraction 3 (see Table 1) on the kinetics of protein synthesis in a reticulocyte lysate supplemented with 20 ,uM hemin. After 5 min of maximal linear synthesis, there is an abrupt cessation of protein synthesis similar to

tor

Inhibitor required to Protein kinase: shut-off pmol 7_32P protein incorporated synthesis -cAMP +cAMP (jig)

+hemin

6____

O

a

1. 2. 3. 4. 5.

S150 extract 0-40% (NH4)2SO4 DEAE-Sephadex Sephadex G-200 DEAE-Sephadex

114.0 4.5 2.0 0.3

1 6 32 72 123

6 72 212 310 517

Protein synthesis in reticulocyte lysates was measured by the uptake of [14C]leucine at 300 for 30 min as described in Materials and Methods. The protein fractions obtained in each step of the purification procedure were titrated to determine the Ag of protein (shown in table) required to reduce [14C]leucine incorporation to the level observed in controls without hemin (see Fig. 1). The protein kinase assay (50 4l) for each fraction was carried out in the presence and absence of 10 MM cAMP as described in Materials and Methods. Incubations were initiated by the separate additions of 11.4 Mg of Fraction 1, 3.3 Mg of Fraction 2, 0.88 Mig of Fraction 3, 0.63 Mg of Fraction 4, or 0.34 Mig of Fraction 5. After 15 min at 300, the extent of [(y-32P]ATP incorporation into histone II was assayed as described. Values represent pmol of 32P incorporated per gg of protein and are corrected for zero time backgrounds (16 pmol/50,Ml); one pmol is 168 cpm.

that induced by heme-deficiency (Fig. 1). The initial period of maximal synthesis in the presence of purified inhibitor indicates that inhibition most likely requires the accumulation or depletion of other components prior to shut-off. Association of a PK Activity with Translational Inhibitor Formed in Heme-Depleted Reticulocyte Lysates. Our recent observation that cAMP reverses the action of the translational inhibitor led to the finding that a highly active PK copurifies with the reticulocyte translational inhibitor (Table 1). The inhibitor has been purified over 300-fold (Table 1). In a 50-Ai reaction mixture supplemented with 20 ,MM hemin, 1060 pmol of [14C]leucine were incorporated in 30 min at 30' compared to 205 pmol of [14C]leucine in the absence of hemin; by comparison, 0.3 ,Ag of Fraction 5 produced a similar degree of inhibition in a hemin-supplemented incubation (Table 1). Each inhibitor protein fraction obtained during purification was assayed for PK activity in the presence and absence of 10 MM cAMP. Table 1 shows that the purification of the inhibitory function was accompanied by a corresponding purification of PK activity. Since reticulocyte S150 extracts contain three known histone receptor protein kinases (24, 25), the extent of purification of the inhibitor-associated PK cannot be determined quantitatively. Some Characteristics of Inhibitor-Associated PK Activity. Calf thymus histone II served as an efficient receptor for y- 32p; there was no detectable autophosphorylation in the absence of histone. The extent of histone phosphorylation was proportional to the amount of inhibitor protein (Fig. 2) and at limiting concentrations was linear with time for at least 1S min (Fig. 2). In the absence of cAMP, there was a lag in the early time course of phosphorylation which may be due to some spontaneous dissociation of the inhibitor-associated PK, resulting in the release of a cAMP-independent catalytic subunit. A comparison of cAMP and cGMP as cofactors for the inhibitor-associated PK activity indicated a requirement for

Biochemistry: LO)

~.50Protein 500

Levin et al.

concentration

Time course

W

~~~~~~~~+cAMP


7

0.2 04 0.6 Q8 1 QO MICROGRAMS PROTEIN

Inhibitor fraction

f

/

E

-lao al.

ITOM CAM/P

24

00/0

I-200-

E

x

0.

CC300

0 z

cGMP = 4

02 0 200

0 a-

4851

half-maximal values 1F M cAMP.4xO

a

X

400-

Cr

Proc. Nat. Acad. Sci. USA 72 (1975)

GTP

utilization

1

4

2

59

2 6

2 10

3

336

10

34

Reaction mixtures (50 Ml) containing 10 AM cAMP were prepared as described in Materials and Methods. Incubation was at 30° for 15 min and was initiated by the addition of 11.4 Ag of Fraction 1, 3.3 Mg of Fraction 2, or 0.9 Mg of Fraction 3 (see Table 1). Values represent pmol of 32p incorporated in 15 min/Mg of inhibitor protein and are corrected for zero time backgrounds (19 pmol/50 Al with labeled ATP, and 13 pmol/50 Al with labeled GTP); one pmol of [-y-32P]ATP is 130 cpm; one pmol of [y-32p]GTP is 187 cpm.

'

FIG. 3. Comparison of cAMP and cGMP as cofactors of the protein kinase activity associated with the reticulocyte inhibitor. Protein kinase activity was assayed in 50-Ml reaction mixtures, and cAMP and cGMP were added as indicated. Reactions were initiated by the addition of 0.34 Ag of inhibitor Fraction 5 (see Table 1) and assayed as described in Materials and Methods. Values are expressed as pmol of [--32P]ATP incorporated in 15 min at 30° and are corrected for zero time backgrounds (15 pmol/50 IsI); one pmol is 108 cpm.

similar preincubation of Fraction 3 in the absence of cAMP resulted in only a slight loss of a cAMP-dependent PK activity and no loss at all of inhibitory function (Table 3). This effect is more clearly demonstrated in the kinetics of PK activity of the two preincubated fractions compared to controls that were not preincubated (Fig. 4). Comparison of Two PK Activities Purified from Hemin-Treated and Heme-Depleted Reticulocyte S150 Extracts. Protein kinases from hemin-supplemented and heme-deficient lysates were partially purified and characterized (Materials and Methods). Chromatography on DEAE-cellulose yielded two PK activities, PK-I and PK-II (Table 4). The PK-II activities isolated from the two lysates displayed different properties. PK-II from the hemin-treated lysate was not cAMP-dependent and had little or no inhibitory activity; PK-II from the heme-depleted lysate displayed significant cAMP-dependency and contained the Table 3. Effect of preincubation of reticulocyte inhibitor in presence and absence of cAMP on PK activity and inhibitory function

Inhibitor treatment

Untreated Preincubated (-cAMP) Preincubated (+cAMP) Not added (-hemin) Not added (+hemin)

Protein kinase: pmol 7y 32p incorporated

Protein synthesis: pmol `[4C] Leu incorporated

375 275 80

230 240 590 145 747

-

-

Aliquots of Fraction 3 inhibitor (see Table 1) were preincubated for 10 min at 340 in the presence and absence of 10 AM cAMP. Protein kinase activity of the inhibitor fractions was measured in the presence of 20 AM cAMP in 50-Ml reaction mixtures. containing 0.9 Mg of the indicated protein sample as described in Materials and Methods. Values are expressed as pmol of [y-32P]ATP incorporated at 300 and are corrected for zero time backgrounds. Protein synthesis was measured as described in Materials and Methods. All incubations contained 20 AM hemin except where indicated; 0.9 Ag of each inhibitor fraction was added at the beginning of incubation. No inhibitor was added to two controls incubated in the presence and absence of hemin.

4852

Proc. Nat. Acad. Sci. USA 72

Biochemistry: Levin et al.

10

20

MINUTES

FIG. 4. Effect of preincubation of translational inhibitor in presence and absence of cAMP on the kinetics of protein kinase activity. Fraction 3 inhibitor (see Table 1) was preincubated with and without cAMP for 10 min at 340 (see Table 4). Protein kinase activity of the treated and untreated inhibitor was measured with time with and without cAMP at 300. Incubations (50 gl) were initiated by the addition of 0.9 Atg of the indicated protein sample, and aliquots were assayed with time as described in Materials and Methods. Values are expressed as pmol Of 32p incorporated per 50 Ml and are corrected for zero time backgrounds (18 pmol/50 ,4; 129 cpm/pmol). Left panel, untreated Fraction 3; middle panel, Fraction 3 preincubated without cAMP; right panel, Fraction 3 preincubated with 10,uM cAMP.

translational inhibitor (Table 4). Hence the correlation between cAMP-dependency and inhibitory function observed with the purified inhibitor (see Table 3) is suggested here as well. It should be noted that in two separate fractionations, no translational inhibitor was found in either PK-I fraction. DISCUSSION Present evidence indicates that the various inhibitions of protein synthesis in reticulocyte lysates induced by hemedeficiency (1, 3, 4), dsRNA (12-16), GSSG (15, 17, 18), or a reticulocyte translational inhibitor (2, 4, 7-9), are exerted through similar mechanisms. Whereas the inhibition in-

duced by heme deficiency is subject to reversal by hemin, Table 4. Comparison of two protein kinase activities isolated from S150 extracts of hemin-treated and heme-deficient lysates

pmol y_32P incorporated

PK I

treatment

+cAMP

-cAMP

activities

+ hemin

29 27 24 62

16 21 26 13

1.8 1.3 0.9 4.8

hemin + hemin

-

II

Ratio of

+cAMP/-cAMP

Lysate

-hemin

(1975)

the other three inhibitions occur in the presence of hemin concentrations that are optimal for protein synthesis. These findings suggest that the event that is regulated by hemin precedes the hemin-insensitive events which are influenced by the addition of dsRNA, GSSG, or the translational inhibitor. The site of hemin regulation remains unknown, but some insight into this problem is provided by the observation that the translational inhibitor contains a cAMP-dependent PK activity. This PK activity was characterized utilizing histone as the phosphate receptor and [,y-32P]ATP as the phosphate donor. Phosphorylation was increased about 6-fold by cAMP. No significant effect on phosphorylation or cAMPdependency was observed by the addition of several components that induce or reverse the inhibition of protein synthesis in lysates, including poly(rI).poly(rC) (20 ng/ml) (16), 2 mM GSSG (15, 17, 18), 2 mM 2-aminopurine (22), and 40 gM hemin. However, since the inhibitor-associated PK activity has been characterized in these studies with histone, a nonspecific phosphate receptor, the possibility should be emphasized that this PK may display different requirements with its endogenous substrate(s). Some indication that the PK activity is associated with the inhibitory function derives from the observation that the preincubation of purified inhibitor in the presence of cAMP produced a significant reduction in both the cAMP-dependent PK and inhibitor activities; a similar preincubation in the absence of cAMP had little or no effect on either function. Inhibition seems to be related to cAMP-dependency. This apparent relationship is also reflected in a parallel study in which we compared two protein kinase activities (PK-I and -II) isolated from both hemin-supplemented and hemedepleted lysates. Based on the chromatographic properties of reticulocyte kinases characterized in other studies, PK-I and -II appear to correspond, respectively, to the peak I and II activities of Tao et al. (23), and the IIH and IIIH activities described by Traugh et al. (25). In the present studies, the PK-II fraction from heme-depleted extracts contained the translational inhibitor and displayed the same cAMP-dependency observed in the purified inhibitor-associated protein kinase. Hence purification of the translational inhibitor and characterization of the reticulocyte protein kinases provide reciprocal confirmation of the relationship between cAMPdependency and inhibitory function. These findings suggest that hemin in some manner regulates the activities of the reticulocyte protein kinases. It should be emphasized that the possibility that hemin affects other sites as well is not precluded. To explain our findings we would like to propose a simple preliminary model which is based on two assumptions, namely that (a) hemin plays a role in altering protein kinase activity, and (b) the inhibitorassociated protein kinase is involved in the inhibitory function.

The model assumes an equilibrium between two endogenous forms of a given PK complex: -hemin

The partial purifications of two protein kinase activities from the ribosome-free S150 extracts of hemin-treated and heme-deficient lysates are described in Materials and Methods; in the final purification step two PK activities were eluted from DEAE-cellulose at 0.1 M KCl (PK-I) and at 0.24 M KCl (PK-II). Protein kinase activity was assayed in the presence and absence of 10 MM cAMP as described; incubations were initiated by the addition of 2-4 Mg of each of the indicated fractions. PK-II of the hemin-deficient extract contains the translational inhibitor described in the text and in Table 1. Values are expressed as pmol Of 32P incorporated per g of protein and are corrected for zero time backgrounds (16 pmol/50 ,l); 1 pmol is 168 cpm.

[PKalnoinhibitory

+cAMP

[PKb]inhibitor

The main features of the model are: (a) In heme-deficiency, the equilibrium strongly favors formation of the PKb complex, which is the translational inhibitor. (b) In hemin-sufficiency, the equilibrium favors formation of the PKa complex, a noninhibitory form which may normally be stabilized by endogenous cAMP. (c) High concentrations of cAMP are required for reversal; PKb formation may be accompanied by a depletion of endogenous cAMP. (d) Hemin

Biochemistry:

Levin et al.

Proc. Nat. Acad. Sci. USA 72 (1975)

regulation may act at one of two sites; the first is directly on the formation of PKb from PKa,

PKa

-hemin

PKb, and the second is on the activation of a cytoplasmic component, X, which then acts to convert PKa to PKb, (i)

-

+ hemin

-

-hemin

(U) Xinactive

-

+ hemin

Xactive

The nature of the translational inhibitor (PKb) is not yet clear. However, it is of interest that a study of reticulocyte protein kinases by Tao and Hackett (24) demonstrates that the regulatory subunit of one molecular species can interact with the catalytic subunit of other protein kinases. Accordingly, it is possible that accumulation of a regulatory subunit might alter the properties of heterologous catalytic subunits. Alternatively, PKb may block protein kinase activity in situ; the fact that the protein kinase activity associated with the PKb complex is not blocked in vitro may be due to some dissociation of the purified complex under the conditions of incubation. This model does not attempt to explain the inhibition by dsRNA or GSSG. At present, this model does not attempt to explain the applicability of this hypothesis to the inhibitions induced by dsRNA or GSSG. On the assumption that the induction of inhibition in heme-deficient lysates involves an altered protein kinase activity, one may speculate on the nature of the endogenous substrate(s). The reversal of inhibition by the initiation factor that mediates the binding of Met-tRNAf to the 40S subunit (11, 15, 21) implicates this factor in the inhibitory mechanism, and suggests the possibility that the factor or a site on the 40S ribosomal subunit associated with the factor is a likely endogenous substrate. In this regard, several reports have described the in vvo and in vitro (25, 28-30) phosphorylation by protein kinases of one or more 40S polypeptides. These observations are consistent with the hypothesis that PK activity may play a role in the regulation of protein chain initiation. This investigation was supported by PHS Grant 7R01 AM16272 and the Harvard-M.I.T. Program in Health Sciences and Technology. D.H.L. is grateful to the Muscular Dystrophy Associations of America for grant support.

Zucker, W. V. & Schulman, H. M. (1968) Proc. Nat. Acad. Sci. USA 59,582-589. 2. Howard, G. A., Adamson, S. D. & Herbert, E. (1970) Biochim. Biophys. Acta 213, 237-243. 3. Maxwell, C. R., Kamper, C. S. & Rabinovitz, M. (1971) J. Mol. Biol. 58, 317-327. 1.

4853

4. Hunt, T., Vanderhoff, G. & London, I. M. (1972) J. Mol. Biol.

66,471-481. 5. Adamson, S. D., Herbert, E. & Kemp, S. F. (1969) J. Mol. Biol. 42, 247-258. 6. Gross, M. & Rabinovitz, M. (1972) Proc. Nat. Acad. Sci. USA 69, 1565-1568. 7. Maxwell, C. R. & Rabinovitz, M. (1969) Biochem. Biophys. Res. Commun. 35,79-85. 8. Rabinovitz, M., Freedman, M. L., Fisher, J. M. & Maxwell, C. R. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 567578. 9. Adamson, S. D., Yau, P. M.-P., Herbert, E. & Zucker, W. V. (1972) J. Mol. Biol. 63,247-264. 10. Gross, M. & Rabinovitz, M. (1973) Biochem. Biophys. Res. Commun. 50,832-838. 11. Ranu, R., Levin, D. H., Clemens, M., Cherbas, L. & London, I. M. (1975) Fed. Proc. 34,621. 12. Ehrenfeld, E. & Hunt, T. (1971) Proc. Nat. Acad. Sci. USA 68, 1075-1078. 13. Hunt, T. & Ehrenfeld, E. (1971) Nature New Biol. 230, 9194. 14. Darnbrough, C., Hunt, T. & Jackson, R. J. (1972) Biochem. Biophys. Res. Commun. 48, 1556-1564. 15. Clemens, M. J., Safer, B., Merrick, W. C., Anderson, W. F. & London, I. M. (1975) Proc. Nat. Acad. Sci. USA 72, 12861290. 16 Hunter, T., Hunt, T. & Jackson, R. J. (1975) J. Biol. Chem.

250,409-417. 17. Kosower, N. S., Vanderhoff, G. A., Benerofe, B., Hunt, T. & Kosower, E. M. (1971) Biochem. Biophys. Res. Commun. 45, 816-821. 18. Kosower, N. S., Vanderhoff, G. A. & Kosower, E. M. (1972) Biochim. Biophys. Acta 272,623-637. 19. Balkow, K., Mizuno, S., Fisher, J. M. & Rabinovitz, M. (1973) Biochim. Biophys. Acta 324,397-409. 20. Legon, S., Jackson, R. J. & Hunt, T. (1973) Nature New Biol. 241, 150-152. 21. Clemens, M. J., Henshaw, E. C., Rahamimoff, H. & London, I. M. (1974) Proc. Nat. Acad. Sci. USA 71, 2946-2950. 22. Legon, S., Brayley, A., Hunt, T. & Jackson, R. J. (1974) Biochem. Biophys. Res. Commun. 56,745-752. 23. Tao, M., Salas, M. L. & Lipmann, F. (1970) Proc. Nat. Acad. Sci. USA. 67,408-414. 24. Tao, M. & Hackett, P. (1973) J. Biol. Chem. 248, 5324-5332. 25. Traugh, J. A., Mumby, M. & Traut, R. R. (1973) Proc. Nat. Acad. Sci. USA. 70,373-376. 26. Traugh, J. A. & Traut, R. R. (1974) J. Biol. Chem. 249, 1207-1212. 27. Rubin, S., Erlichman, J. & Rosen, 0. M. (1972) J. Biol. Chem. 247,36-44. 28. Kabat, D. (1971) Biochemistry 10, 197-203. 29. Gressner, A. M. & Wool, I. G. (1974) J. Biol. Chem. 249,

6917-6925. 30. Ashby, C. D. & Roberts, S. (1975) J. Biol. Chem. 250, 25462555.