Nature of Ribosome-Bound f-galactosidase

Transcription

1 JOURNAL OF BACTRIOLOGY, Feb. 1971, p Vol. I05. No. 2 Copyright A 1971 American Society for Microbiology Printed in U.S.A. Nature of Ribosome-Bound f-galactosidase PATRICIA M. BRONSKILL AND J. TZ-FI WONG Department of Biochemistry, University of Toronto, Toronto 5, Ontario, Canada Received for publication 31 July 1970 Properties of the ribosome-bound,b-galactosidase were examined in scherichia coli cells after prolonged induction. This fraction of enzyme was not chased from ribosomes by removal of inducer, or by treatments with hydroxylamine, puromycin, chloramphenicol, and azide. However, the metabolic turnover of this fraction could be demonstrated by means of a pulsed exposure to the phenylalanine analogue d-2- thienylalanine, and this fraction was enriched in heavy forms relative to the soluble enzyme. These observations indicated a tight coupling of the release of ribosomebound enzyme to nascent enzyme synthesis, and it is suggested that the ribosomebound enzyme is related to an intermediate stage in the assembly of quarternary enzyme structures. The existence of ribosome-bound fl-galactosidase in scherichia coli is well established (2, 5, 14). Moreover, on the basis of their biosynthetic behavior, Zipser has distinguished between two separate fractions of the ribosome-bound enzyme. A chaseable fraction appeared rapidly upon induction, and disappeared after the removal of inducer. A nonchaseable fraction accumulated gradually, and became predominant at later stages of induction. Since it was not chased from ribosomes after the removal of inducer, this fraction had been tentatively interpreted as being translational "failures" which became stuck to the site of biosynthesis. Since this interpretation requires a high failure rate in translation and since, in general, the release of newly synthesized proteins from ribosomes is poorly understood, this study attempted to examine further the nature of this nonchaseable fraction of fb-galactosidase. MATRIALS AND MTHODS Bacteria and reagents. xperiments were carried out with. coli strain ML3 lac i+z+y- (ATCC 15223). Cells were grown in mineral medium "63" (12) with 1% glycerol as the carbon source. lsopropyl- 3-D-thiogalactopyranoside (IPTG), o-nitrophenyl-fl-d-galactopyranoside (ONPG), and DL-,8-2- thienylalanine were purchased from Mann Research Laboratories; thienylalanine-3-'4c was obtained from Calbiochem, and Sephadex G-200, from Pharmacia. TM buffer used was 10-2 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride and 10-2 M magnesium acetate, ph 7.3. Preparation of soluble and ribosome-bound,b-galactosidase. Populations in exponential growth were induced with 0.4 mm IPTG for I hr. The cells were centrifuged and washed once with cold TM buffer. The pellet was resuspended in TM buffer and put through a 498 French hydraulic press. Deoxyribonuclease was added to 3 mg/liter, and the suspension was centrifuged twice at 30,000 x g for 15 min to give the crude extract. The crude extract was centrifuged at 50,000 rev/min (Spinco no. 50 rotor) for 45 min, and the supernatant fluid contained the soluble enzyme. The pellet was resuspended in TM buffer by gentle homogenization, and centrifugation was repeated three more times to obtain the ribosome-bound enzyme. All operations were at close to 0 C. Ribosome concentration was determined by absorbancy at 260 nm, on the basis of 66,g of ribosomes per ml per unit of absorbancy. The soluble enzyme was purified as described by Craven, Steers, and Anfinsen (3). Of the three alternative steps described by these authors to follow chromatography on Sephadex G-200, density gradient centrifugation was chosen in view of the small amount of enzyme handled. The ribosomal enzyme was removed from ribosomes by treatment with sodium dodecyl sulfate and spermine (14). Density gradient centrifugation of either soluble or stripped ribosomal enzyme was performed by layering on a gradient of 5 to 20% sucrose in TM buffer and centrifuging for 16 hr at 28,000 rev/min in a Spinco SW41 rotor. The fractions, collected by puncturing the tube and counting drops, were assayed for enzymatic activity. When radioactivity also was to be determined, the fractions were precipitated by 5% trichloroacetic acid in the presence of mg of bovine serum albumin as a carrier, washed on a membrane filter with 0.45-jm pores, and finally counted in a Nuclear-Chicago low-background gas-flow counter with 22% efficiency for 14C. Assay for enzymatic activity. To assay suspensions of whole cells, a l-ml sample was mixed with 0.05 ml of toluene and continually inverted on a rotating rack for I hr. The activity of,b-galactosidase was determined by the procedure of Pardee, Jacob, and Monod (10), with one unit of enzyme activity being that amount which hydrolyzes I nmole of ONPG per min. The same assay was used for ribosomal suspensions, but samples were first dispersed by sonic treatment. To test the stability of enzyme preparations in urea,

2 VOL. 105, 1971 RIBOSOM-BOUND fb-galactosidas 499 the sample was incubated for various times at 37 C with 3 M urea, 0.01 M Tris-hydrochloride, ph 7.4, 0.01 M magnesium acetate, 0.1 mm manganese chloride, 0.1% mercaptoethanol, and 0.3% bovine serum albumin. At the end of incubation, a sample was diluted 20-fold with TM buffer and assayed for enzymatic activity. RSULTS Properties of ribosome-bound l-galactosidase. Between three and six centrifugal washes, the enzymatic activities bound to ribosomes remained at a constant level of 0.10 unit/ug of ribosomes for cells induced for I hr, or 0.02 unit/,g of ribosomes for uninduced cells. The addition of soluble enzyme from induced cells to an extract from uninduced cells failed to enhance the final level of ribosomal activity, thus ruling out physical adsorption as the origin of ribosomebound enzyme. The association between enzyme and ribosomes was also demonstrated by density gradient sedimentation and by a precipitation of 97% of the activity by spermine. These observations confirmed the previous reports of Cowie et al. (2), Zipser (14), and Fogel and lson (5). After being stripped from the ribosomes by means of sodium dodecyl sulfate and spermine, the bulk of the enzyme resembled the soluble enzyme in sedimentation, but there was an evident enrichment of heavier forms in this fraction (Fig. 1). ffects of terminations of enzyme synthesis. As U N ILl x a 50 shown in Fig. 2, the induced synthesis of,b-galactosidase in whole cells could be terminated by a number of treatments. None of these treatments was effective in reducing the level of enzymatic activity in the ribosomes (Table 1). The enzyme was not extensively chased from ribosomes by hydroxylamine, chloramphenicol, azide, or removal of inducer itself. However, considerable chasing was brought about by the phenylalanine analogue thienylalanine, which decreased but did not terminate the rate of active enzyme synthesis by the cells. Turnover of ribosome-bound,3-galactosidase. Since the ribosomal activity was reduced by a short exposure to thienylalanine, an active turnover of this fraction was indicated. This possibility was tested further by means of the greater lability toward denaturation by urea of,b-galactosidase synthesized in the presence of thienylalanine (8). For this purpose, three types of cell preparations were compared: (i) cells induced normally for I hr, (ii) cells induced in the presence of thienylalanine for 4 hr, and (iii) cells induced normally for I hr and then exposed to a 10-min pulse of thienylalanine. In the pulsed cells, the soluble enzyme resembled the enzyme of normal cells in lability toward urea, but the ribosome-bound enzyme resembled that of cells grown for 4 hr in the presence of thienylalanine (Fig. 3). Since exposure to a pulse of thienylalanine did not bring about an overall increase in Fraction Number FIG. 1. Comparison between soluble enzyme and enzyme stripped from ribosomes. nzyme preparations were obtained from cells induced for I hr. Sodium dodecyl sulfate and spermine were added to the soluble enzyme to render the solution similar in composition to that of the stripped enzyme. ach enzyme solution was centrifuged through a S to 20% sucrose gradient for 16 hr at 28,000 rev/min in an SW 41 rotor. Solid line, soluble enzyme; dashed line, stripped enzyme.

3 500 BRONSKILL AND WONG J. BACTRIOL.. 50 N C0 X- AU 0 (b), (c) (d) (6) 1_-5g= i a. (f -- (9) a FIG. 2. ffects of various treatments on cellular synthesis of,8-galactosidase. In all cases, cells had been induced for I hr before treatment was started, and activities were normalized for a cell suspension of 0.4 optical density at 450 nm in a Gilford spectrophotometer. (a) Control cells with normal induction; (b) inducer removed by two centrifugations; (c) addition of 50 Mm hydroxylamine; (d) addition of I mm thienylalanine; (e) addition of 20 mm sodium azide; (I) addition of88 Mg ofpuromycin per ml in magnesium-free medium, according to Sells (11); (g) addition of Mg ofchloramphenicol per ml. ribosomal activity (Table 1), these results point to an extensive replacement of the normal enzyme on the ribosomes by enzyme containing the analogue. To demonstrate the incorporation of thienylalanine into,b-galactosidase, 14C-thienylalanine was employed, and the soluble enzyme was purified by the procedure of Craven, Steers, and An- TABL 1. ffects ofdifferent treatments on the level of ribosome-bound,8-galactosidase Treatment Relative specific activity of ribosome-bound,8-galactosidasea Control Removal of IPTG Hydroxylamine, 50 Mm (IPTG present) Hydroxylamine, 50 um (IPTG removed) Puromycin, 88 Mg/ml Sodium azide, 20 mm Chloramphenicol, O,g/ml Thienylalanine, I mm a For measurements of ribosome-bound activities, cells were harvested after 15 min of each type of treatment. Specific activities of units of enzyme per microgram of ribosomes are expressed relative to control cells without any treatment. finsen (3). After the final step of density sedimentation, a well-defined peak of radioactivity was coincident with the enzymatic activities (Fig. 4). By use of the specific activity and amino acid composition for galactosidase reported by these authors, the peak fraction from the gradient was calculated to contain 4.1 nmoles of W-thienylalanine in 12 Ag of enzyme. This corresponds to 107% replacement of phenylalanine by analogue. Although it was necessary to correct for inhibition by sucrose from the gradient as well as for the different absorbancies of o-nitrophenol at different ph, the data from Fig. 4 sufficed to indicate an efficient replacement of phenylalanine by the analogue. DISCUSSION In this study, the level of ribosomal f-galactosidase activity in. coli cells after prolonged induction was observed to be undiminished by the removal of inducer. This agrees with the findings of Duerksen and Cowie (4) and conforms to the nonchaseable fraction described by Zipser (14). Since ribosomes are the site of enzyme synthesis, it might be expected that enzyme molecules upon completion of translation will be chased from the ribosomes into the soluble fraction of the cell. At least several mechanisms can be recognized, how- I 9 f)

4 VOL. 105, 1971 RIBOSOM -BOUND,-GALACTOSIDAS FIG. 3. Denaturation of fi-galactosidase by 3 M urea. (i) Soluble enzyme; (ii) ribosome-bound enzyme; (iii) ribosome-bound enzyme after removal from ribosome by sodium dodecyl sulfate and spermine. In all these cases, curve a represents preparation from cells normally induced for I hr, curve b, preparation from cells induced in the presence of I mm thienylalanine for 4 hr, and curve c, preparation from cells induced normally for I hr and then exposed to thienylalanine for 10 min. 05 c-~ LU C0 CD ; I'is 0> a- 400 'j Fraction Number FIG. 4. Incorporation of,3-thienylalanine into il-galactosidase. Soluble enzyme was prepared from 500 ml of a cell suspension which had been induced for 4 hr in the presence of I mm [14C]-D, L-thienylalanine (0.1 uci/,mole). After ammonium sulfate fractionation and chromatography on Sephadex G-200, the enzyme was concentrated by ammonium sulfate precipitation and dialyzed before being subjected to density gradient centrifugation. Determinations of the enzymatic activity and radioactivity of the gradient fractions were as described in Materials and Methods. Solid line, enzyme activity; dashed line, radioactivity. ever, which might account for a lack of chasing of ribosomal,b-galactosidase. (i) The presence of inducer may be required for the release of enzyme from ribosmes; such a requirement indeed has been discussed as a plausible mode of inducer action in controlling enzyme synthesis (7, 13). To test this possibility, enzyme synthesis was terminated with hydroxylamine. Since hydroxylamine had been demonstrated to inhibit the initiation but not the completion of enzyme synthesis (9), its failure to chase the enzyme from the ribosomes even in the

5 502 BRONSKILL AND WONG J. BACTRIOL.. presence of inducer (Table 1) rules against such an inducer-dependent release mechanism. (ii) Zipser suggested that the ribosomal enzyme might represent translational failures which become stuck to the ribosomes, but pointed out that such failures would lead to an unacceptably high rate of ribosome inactivation unless the growing enzyme molecule can exhibit full activity even when it is far from completion. Also, such failures would be capable of metabolic turnover only if some elimination mechanism operates to remove them continuously from the ribosomes. Several lines of evidence in this study are incompatible with either of these two requirements. First, the lack of chasing by puromycin (Table 1) rules out any binding of translational failures to the ribosomes through transfer ribonucleic acid (6). Second, ribosomal enzyme was undiminished 15 min after enzyme synthesis was halted by either chloramphenicol or azide (Table 1), and therefore no elimination mechanism removing the enzyme from the ribosomes was apparent. Finally, Zipser had found the stripped ribosomal enzyme to be indistinguishable from the soluble enzyme in sedimentation, but it was not specified whether the experiment was performed with the early chaseable fraction or the later nonchaseable one. In Fig. 1, the nonchaseable enzyme was stripped from ribosomes and examined. Its main component was similar to the soluble enzyme in sedimentation, but there was an evident enrichment in heavier forms which migrated ahead of the main component. Thus, the ribosomal activity was derived from enzyme molecules with a full complement of quarternary structures rather than from translational failures which were far from completion in chain length. (iii) When induced cells were exposed to a short pulse of thienylalanine, the soluble enzyme in the cells remained normal in its lability towards urea, but the ribosomal enzyme largely exhibited the lability typical of the thienylalanine enzyme (Fig. 3). Since the incorporation of thienylalanine into f3-galactosidase could be demonstrated directly (Fig. 4, and reference 8), the lability studies suggest an active metabolic turnover of the ribosomal enzyme. [Interestingly, the different behavior responses of the soluble and ribosomal enzymes reported in Fig. 3 also rule out the ribosomal enzyme being an adsorption artifact formed at the instant of cell breakage, a possibility discussed by Duerksen and Cowie (4).] This turnover of the ribosome-bound enzyme and its lack of chasing by various treatments to terminate enzyme synthesis together point to a tight coupling between its turnover and a continual synthesis of nascent enzyme molecules. A simple basis for such a coupling can be represented as follows: nascent subunit ribosome-bound enzyme + n soluble enzyme Although the detailed mechanisms for assembling the quarternary structures of oligomeric enzymes are obscure at present, it is evident that a nascent enzyme subunit needs to react with suitably receptive components in order to be incorporated into quarternary aggregates. The in vitro study of Zipser and Perrin (15) already suggested that such receptive components may be ribosomebound. If such is the case in vivo, the metabolic turnover of receptive components on the ribosomes would become dependent on the production of nascent enzyme subunits, as depicted in the above scheme. The source of the nascent subunit reacting with the ribosome-bound enzyme remains unspecified; it may be newly synthesized on the same ribosome to which the enzyme is bound, or it may come from another ribosome. This proposal that the ribosomal f-galactosidase is associated with an intermediate stage in the process of quarternary assembly is compatible with additional lines of observation. Zipser and Perrin (15) reported an exceptionally high fraction of the enzyme bound to the ribosomes in a mutant z, strain of cells, and structural mutations in protein molecules are known to be capable of affecting their quarternary interactions. Appel, Alpers, and Tomkins (1) also observed upon enzyme induction an accumulation of heavy forms of the enzyme which sedimented ahead of the main 16S component. Thus, the heavy forms are apparently related to the increased rates of enzyme synthesis and assembly, and from Fig. 1 the ribosomal enzyme is seen to be particularly enriched in heavy forms. At present, elucidation of the detailed mechanism for the quarternary assembly of,b-galactosidase must await further experiments. The observations in this study on ribosomal,b-galactosidase indicate its involvement in this mechanism, and a search for a similarly ribosome-bound, nonchaseable fraction among other oligomeric enzymes would be of obvious interest. ACKNOWLDGMNTS This study was supported by the Medical Research Council of Canada. and one of us (P.M.B.) held a Medical Research Council Studentship. We are also indebted to Mrs. Alix Pincock for invaluable assistance and A. T. Matheson for helpful comments. LITRATUR CITD 1. Appel, S. H., D. H. Alpers, and G. M. Tomkins Multiple molecular forms of,t-galactosidase. J. Mol. Biol. 11: Cowie, D. B., S. Spiegelman, R. B. Roberts, and J. K. Duerksen Ribosome-bound 6-galactosidase. Proc. Nat. Acad. Sci. U.S.A. 47: Craven, G. R., J.. Steers, Jr., and C. B. Anfinsen Purilcation, composition and molecular weight of the 0-galactosidase of scherichia coli K12. J. Biol. Chem. 240:

6 VOL. 105, 1971 RIBOSOM-BOUND O-GALACTOSIDAS Duerksen, J. D., and D. B. Cowie Ribosomal enzymes. Carnegie Inst. Wash. Year B. 60: Fogel, Z., and D. lson The ribosomal #-galactosidase of scherichia coli. Biochim. Biophys. Acta 80: Gilbert, W Polypeptide synthesis in scherichia coli. I. The polypeptide chain and s-rna. J. Mol. Biol. 6: Harris, H Nucleus and cytoplasm, p. 71. Clarendon Press, Clarendon, Tex. 8. Janecek, J., and H. V. Rickenberg The incorporation of js-2- thienylalanine into the,8-galactosidase of scherichia coli. Biochim. Biophys. Acta 81: Kepes, A., and S. Beguin Hydroxylamine, an inhibitor of peptide chain initiation. Biochem. Biophys. Res. Commun. 18: Pardee, A. B., F. Jacob, and J. Monod The genetic control and cytoplasmic expression of "inducibility" in the synthesis of,-galactosidase by. coli. J. Mol. Biol. 1: Sells, B. H RNA synthesis and ribosome production in puromycin-treated cells. Biochim. Biophys. Acta 80: Sistrom, W. R On the physical state of the intracellularly accumulated substrates of #-galactoside-permease in scherichia coli. Biochim. Biophys. Acta 29: Vogel, H. J Repression and induction as control mechanisms of enzyme biogenesis, p In W. D. Mclroy and B. Glass (ed.), The chemical basis of heredity. Johns Hopkins Press, Baltimore. 14. Zipser, D Studies on the ribosome-bound,b-galactosidase of scherichia coli. J. Mol. Biol. 7: Zipser, D., and D. Perrin Complementation on ribosomes. Cold Spring Harbor Symp. Quant. Biol. 28: Downloaded from on October 26, 2018 by guest