Reprinted from the Proceedings of the Narionau ACADEMY OF SCIENCES Vol. 47, No. 1, pp. 114-122. January, 1961. RIBOSOME-BOUND 6-GALACTOSIDASE By D. B. Cowin, 8. Sprecetman,* R. B. Roperts, ano J. D. DuEeRKsuNt DEPARTMENT OF TERRESTRIAL MAGNETISM, CARNEGIE INSTITUTION OF WASHINGTON, D. C., AND DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA Communicated by M.A. Tuve, October 20, 1960 Considerable evidence has been accumulated to show that ribonucleoprotein particles (ribosomes) provide sites for protein synthesis in animal cells.1 Studies of the incorporation of radioactive tracers by Escherichia coli also indicated that the ribosomes of bacteria are active in protein synthesis (McQuillen, Roberts, and Britten’). These authors showed that in growing bacteria, a quantity of material roughly equal to the protein synthesized in three seconds was transiently associated with the ribosomes before being released to the soluble protein fraction of the cell. Since the rate of protein synthesis in LZ. cold is about 0.02 per cent per second, 0.06 per cent of any particular protein might be expected to be found transiently associ- ated with the ribosomes. A series of experiments was started to determine whether B-galactosidase showed the same transient association with ribosomes as was indicated for proteins in general. The first experiments showed that a small frac- tion of the enzyme was bound to the ribosomes, and it is the purpose of the present paper to describe some of the properties of this ribosome-associated enzyme. Furthermore, the results suggest a general procedure for isolating specific ribosomes. Materials and Methods.—FE. coli strains: Three strains of FZ. cold differing in their 6-galactosidase-synthesizing properties were used, namely ML 30 (inducible), ML 308 (constitutive), and W2214 (absolute negative). Growth conditions: Cells were grown at 37°C in a vigorously aerated synthetic medium (C) of the following composition: 2 g NH.Cl, 6 g Nas HPOu, 3 g KH.PO,, 3g NaCl, 0.01 g Mg as MgCl, 0.026 g 8 as Na.SO,, 900 ml H.O, and 10 ml. of 10 per cent maltose. Enzyme induction and assay: Thiomethyl-6-D-galactoside (TMG) or thioiso- propy]-8-D-galactoside (TIPG) at 5 & 10-4 M were uscd as inducers of 8-galacto- sidase synthesis. Assays of 8-galactosidase on ribosomal preparations were per- formed with the following mixture buffered at pH 7.4; 0.0027 M ortho-nitrophenyl- G-D-galactoside (ONPG), 0.05 47 NaCl, 0.01 M trishydroxymethylaminomethane Vor, 47, 1961 MICROBIOLOGY: COWIE ET AL. 115 (Tris), 0.004 M succinic acid, and 0.01 Af magnesium acetate. The hydrolysis of the ONPG was followed in a Beckman spectrophotometer at 420 mu. The sodium chloride, tris-succinate mixture was used in place of the more commonly employed phosphate buffer because of the known! instability of ribosomes in the presence of phosphate. The rate of ONPG hydrolysis is the same in the two buffer systems. Preparation of wall-free cell juice: Exponentially growing cultures of E. coli were harvested and washed once in a Tris buffer adjusted to pH 7.6 containing 0.01 AY Tris, 0.004 M succinic acid, and 0.01 47 magnesium acetate (TSM). Fol- lowing the wash, the cells were resuspended in 10 ml of the same buffer. The cells c ro} 10} ML-308 (Lac*) F , Ribosomes in | ML-308 12 12 Supernate 7 °o rT [ ° I J 1 3 “4 ' 7 8 &-Galactosidase ty T 2 | W221/4 (Lac™) Ribosomes in ML-308 x Supernate UV poce---s OPTICAL DENSITY AT 260mz ( ENZYMIC ACTIVITY (-~-—-—) SPECIFIC ENZYME ACTIVITY (DIVISIONS/MIN./0.D.260) 4 44 Oli \ A [ \ mo \ 2 2 \ 7 \ 5 | L \ | 1 . 0 2 4 6 0 10 20 30 NUMBER OF WASHES FRACTION NUMBER Fic. 1.—The effect of successive washings by centrifugation on the specific enzyme activ- ity of ribosomes from genetic positives ( @) and ribosomes from genetic negatives (X), both in- itially suspended in an extract containing large amounts of active @-galactosidase. Fic. 2.—Sedimentation pattern of Lac? ri- bosomes washed three times prior to being lay- ered on the sucrose gradient (3 to 20 per cent). The run was made at 37K for 80 min. of this suspension were ruptured by extrusion through a small orifice under pressure (approximately 15,000 Ib/sq. inch) in a modified French pressure cell. The ex- truded material was centrifuged for five minutes at 40,000 rpm in the angle head rotor of the Spinco Model L centrifuge to remove whole cells, cell walls, and other large fragments. Antisera: @-galactosidase antiserum was prepared by injecting rabbits with 10 mg of purified (90 per cent) H. coli 8-galactosidase. Chicken anti-rabbit serum was kindly furnished us by Dr. Alan Boyden, Rutgers University, New Brunswick, N. J. Results Enzymatic activity of ribosomes: Centrifugation of a wall-free cell 116 MICROBIOLOGY: COWIE ET AL, Proc. N. A. S. juice for 45 minutes at 40,000 rpm in the angle head rotor of the Spinco Model L centrifuge gives a pellet (40K 45P) which contains more than 90 per cent of the 708 and 85S ribosomes of the cell. Such a centrifugation leads to a useful separation of the major ribosome components from the bulk of the soluble proteins and smaller particles and yields material suitable for further purification. One technique for purifying large quantities of 855 and 70S ribosomes involves repeated washing in TSM. Figure 1 compares the amounts of enzymic activity found in the pellet fractions (40K 45P) in successive washings of ribosomes derived from ML 308, the constitutive mutant (circles). It will be noted that by the fourth washing a constant specific enzymic activity (enzyme units per O.D. at 260 my) is achieved. 420 s 1 7 5 _ {80 a. 1 -300t- ky ve Galactosidase Ee. 16 | ms 4 =~ ° — ~ | 1 oo aL rm ' | N > Ee r I bE EF o36 \ 1.60 ! wo ' ! oar “22 8 4 io > oO EK -200:- z Ee zt < > Z Q > 405 i 08= §& 9 =! NG 2 3 | 5 2.100 | 2 a 0.4 z -20N oO | oO uJ | = a t o UH | Lo Oo 10 20 30 0 10 20 30 FRACTION NUMBER FRACTION NUMBER Fie. 3.—Sedimentation pattern of the ribo- Fie. 4.—Sedimentation pattern of the ribo- somes in the first 13 fractions of Figure 2. somes in the first 13 fractions of Figure The numbers denote per cent increases in en- 3. zyme activity when the corresponding frac- tion was incubated with anti-6-galactosidase serum. Such data do not necessarily establish a specific association between a fraction of the enzyme molecules and the ribosomes. For example, some enzyme molecules could be nonspecifically but firmly adsorbed to the surface of the ribosomes. Alter- natively, the enzymic activity of the pellet may not be associated with the ribosomes but may reside in aggregates possessing an average sedimentation constant roughly equivalent to the larger ribosomes. The first of these possibilities was excluded by the following experiment. An extract rich in enzyme but free of ribosomes was prepared from the constitutive strain, ML 308 (Lact), by centrifuging the cell juice for five hours at 40,000 rpm. Purified ribosomes prepared from the absolute lactose-negative strain W2214 (Lac—) were suspended in this extract. The resulting mixture was subjected to the Vou. 47, 1961 MICROBIOLOGY: COWIE ET AL. 117 successive centrifugations in TSM. Figure 1 shows that the enzyme associated with the genetically negative ribosomal pellet behaves quite differently from that observed with genetically competent material. Initially, the specific enzymic activities of the two ribosomal pellets are about equal. In the lower curve, there is no suggestion of an approach to a constant specific activity on repeated washings. By the fourth step, the Lac~ ribosomes reach a specific activity (not indicated on the graph) which is less than 0.001 units of enzyme per optical density unit. Such enzymic activities are too low for accurate measurement and correspond to levels considerably less than 1/100 of the constant specific activities attained with the Lact ribosomes. Centrifugation of a layer of ribosome suspension through a sucrose gradient in the swinging bucket rotor of the Spinco centrifuge‘ provides a much better indication that the enzyme is actually associated with the ribosomes. Figure 2 shows the distribution of the enzymic activity and optical density of a ribosome preparation (subjected first to two washes as described above) after being layered on the sucrose gradient and centrifuged for 45 minutes at 37,000 rpm. The enzyme is distributed roughly equally between the ribosomal and supernatant fractions. Figures 3 and 4 illustrate how successive sedimentations through sucrose gradients lead to the elimination of the soluble enzyme component from the ribosome fraction. The first 13 fractions of the run described in Figure 2 were pooled and the ribosomes collected by centrifugation (40K 45P). They were then resuspended and layered on a new sucrose gradient and centrifuged. The resulting profile of O.D. at 260 mu and of enzyme activity is given in Figure 3. Comparatively little enzyme is found unassociated with the ribosome peak although there is clearly a contamination of free enzyme. A repetition of this procedure on the first 13 fractions of Figure 3 is shown in Figure 4. Here, there is an excellent correlation of enzyme activity with ribosome peaks in the density gradient. Nature of the ribosome-associated enzyme: ‘The sedimentation patterns described above encouraged the belief that some enzyme molecules are in physical association with the larger ribosome particles. It was of interest to look for some feature other than the sedimentation characteristics which would serve to distinguish these molecules from those which are in the soluble fraction. A series of experiments were, therefore, performed examining the effect of a variety of agents on the residual ribosomal enzyme activity. Methods of disrupting ribosomes (RNAase, versene, or citrate) which have been shown to be capable of releasing latent RN Aase activity of the ribosomes of £. coli ® did not cause any increase in the 6-galactosidase activity of highly purified ribosomal preparations. Several attempts to activate such preparations by the addition of the galactoside inducers, TMG and TIPG, were also unsuccessful. The effect of adding specific antiserum was examined with the hope that it might serve as a specific means for removing the associated enzyme from the ribosomes. Table 1 shows that exposure of purified ribosomes to anti-G-galactosidase results in a striking increase in enzymic activity. In a series of similar experiments using com- parably purified ribosomes, increases of between four- and sixfold were often observed. This activation is unique for the specific anti-6-galactosidase serum; normal rabbit serum and nonhomologous antisera (e.g. anti-alkaline phosphatase) have no effect. Further, the increase in activity is confined to ribosome-bound 118 MICROBIOLOGY: COWIE ET AL, Proc. N. A.S. TABLE 1 Lirrect oF RaBBiT SERUM UPON RIBOsOME ASSOCIATED AND SOLUBLE §-GALACTOSIDASE Fraction Rabbit serum added Enzyme activity Ribosomes (uninduced ML 30) 0 0.04 Ribosomes ( “ “ey Anti-8-galactosidase 0.15 Ribosomes ( “ a) Normal 0.05 Soluble ( “ “ey 0 0.092 Soluble ( “ “ey Anti-8-galactosidase 0.090 Ribosome fractions were purified by successive centrifugations to constant specific activity and enzyme was assayed with ONPG according to the procedures described under Methods. Soluble enzyme was obtained by removal of ribosomes by means of a sucrose gradient swinging bucket centrifugation. Assays were continued until linear rates were well established. enzyme and is not observed with the soluble enzyme as shown in Figure 3. Aliquots taken from fractions corresponding to different portions of this O.D. profile were tested for their ability to be activated by the specific 6-galactosidase antiserum. The increases over controls are recorded in Figure 3 over the appropriate parts of the profile.t Fractions corresponding to the ribosome region exhibit the antiserum- activating effect. As one proceeds to fractions closer to the free soluble region, however, the degree of activation falls and finally becomes zero. Thus, the soluble enzyme and the enzyme associated with the ribosomes differ not only in their sedimentation rates but also in their response to antibody. Two other possible explanations for such activation are (1) that the addition of ribosomes activates in some manner 6-galactosidase present in the antiserum which is not detectable in their absence or (2) that the presence of ribosomes nonspecifically inhibits the activity of @-galactosidase and this inhibition can be reversed with specific antiserum. ‘To test the validity of the first suggestion, purified ribosomes prepared from the Lac~ strain (W2214) were incubated with the anti-6-galacto- sidase serum. These mixtures were assayed for enzymic activity and none was found. It is concluded from such experiments that the observed increase in activity is not due to a latent enzyme in the antiserum. To examine the second possibility, the following experiment was performed.