Fate of specific ribonucleoside triphosphatases purification and reconstitution of ribosomal system of Escherichia col

Fiala, J.

Raacke, I. D.

English

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I 
FATE OF SPECIFIC RIBOIWCLEOSIDE. TRIPIQSP?W!ASES ON EVRIFICATION 
AND RECONSTITUTION OF THE RIBOSOMAL SYSTEM OF ESCBERICEIA 
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I. D. Raacke and J. Fiala 
Ksiser p'ouadstion Research Institute, Iaboratory of Comparative 
Biology, Richnd, and Space Sciences Laboratory, University of 
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SUMMARY Hard copy (HC) 
xoo WYOd 1 1 1 7 m v ~  - Microficb IM F) i . ? A study was made of the relative(chmge6 in RNA, protein, and- 
of E. coli jm-- L A q  specific triphosphatases 3-- 
Into ribosomal and supernatant fractions1 and when the purified 
ribosomes are subsequently recombined with "supernatant" 
results were obtained: 
The following 
1. In the course of the centrifigal purification of the ribosomal 
system there is a net and irreversible loss of material f r o m  the "heavy" 
and "light" particle fractions, the loss of protein being relatively 
greater than that of RNA. 
2 .  Some, but not all of the protein lost from the ribosomes in the 
course of their purification is readsorbed when washed ribosomes are mixed 
wlth supernatant. 
3. Both the protein and the RRA lost in the washings are qualitatively 
different from that remaining in either the ribosomes or the supernatant. 
%me, the reconstituted system is not equivalent to the crude extract. 
https://ntrs.nasa.gov/search.jsp?R=19650019884 2020-03-17T01:58:03+00:00Z
- 2 -  . 
4. The four triphosphatase activities are differentially removed 
from the ribosomes during the washing procedure. Specifically, washing 
caused an increase in specific activity of UTFbse, a partial 10138 of 
ATPase, a complete, but reversible loss of GTPase, and a complete and 
Irreversible lose of CTPase. The results prove that four different 
enzymes are responsible for the four triphosphatase activities. 
INTIiOIlUCTION 
The existence of specific ribonucleoside triphosphatases in crude 
extracts of E. coli was demonstrated in the preceding paper (1). Since -- 
the bulk of these enzymes appeared to be particle-bound, it seemed of 
interest to Investigate their fate in the course of routine purification 
of ribosomal and supernatant fractions, as commonly employed for studies 
of amino acid incorporation. This is the subject of the present communication. 
All methods of preparation and analysis were a6 described in the two 
preceding papers (1, 2), except that all samples, as well as the sucrose 
density gradients (SEG) were prepared and kept in 0.01 M Mg acetate, 
0.005 M Tris, pH 7.1. This lower pH was chosen to facilitate the adsorption 
t o  the ribosomal fractions of total protein (2), though not necessarily of 
triphosphatases (1). 
An aliquot of crude extract was centrifuged on a SDG, and analyzed 
while ribosomes and supernatant were being prepared from the same extract. 
(The ribosomes were taken through three cycles of centrifugation.) The 
next day ribosomes and supernatant fraction were mixed in a proportion 
approximating that of the crude extract; this reconstituted system was 
then compared with ribosomes and supernatant fraction alone. 
c 
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l3SUIiTS 
The distributions of RNA and protein in the isolated fractions and 
reconstituted system, as compared to that in the crude extract, are given 
I n  Fiw. 1 and 2. 1 It is seen that,’protein and RNA were lost mainly from 
the heavy and light particle fractions, a s  expected from the centrif’ugation 
schehles employed in the washing of the ribosomes. The relative loss of 
protein, however, was greater than that of RNA, as shown by the increased - _- 
RNA to protein ratio of the ribosomes, as compared to the crude extract 
(Fig. 3) .  The RlU to protein ratio profile of the reconstituted system 
was remarkably similar to that of the crude extract, as some, though not 
all, of the protein lost during purification was readsorbed when ribosomes 
were mixed with supernatant, 
As seen in the preceding paper (1) the bulk of the specific triphosphatase 
activities is sedimentable in the crude extracts, and hence appears in the 
crude ribosomal pellets. 
these activities are differentially lost- 
In the process of washing the ribosomes, however, 
CTPase was the most labile, it 
being removed completely in the first wash. Next in lability was GTPase 
which was very l o w  after the second wash. ATPase activity was also reduced 
by washing, but the ultimate level of activity in the purified ribosomes 
varied greatly with both the conditions of purification, as well as the 
nature of a given preparation. (The ribosomes in the present experiment 
had a rather higher activity than usual. ) The UTPase activity was most 
refractory to removal by washing, and a l l  ribosomal preparations examined 
to date contained this activity. Under the particular conditions of the 
present experiment, the specific UTPase activity was actually greater in 
the purified ribosomes than in the crude ones. Fig. 4 shows the distribution 
Of UTPase activity in a crude ribosomal >ellet (which, however, was low 
- 4  - 
in ATPase activity), and Fig. 5 shows the distribution of UTPase and 
ATPase in the ribosomes used in the present experiment. 
was free of CTPase, and essentially free of GTPase activity.) 
(This preparation 
It should 
be noted that the enzyme peaks in the purified ribosomes are as sharp or 
sharper than in the crude extract, showing that ribosomes remain hetero- 
geneous even' after purification (see also (2)). 
4 
Also, the specific activity 
of the enzymes is highest in the heavy regions of the gradient, just as 
in crude extracts (1) and very l o w  in the main peak. 
ribosomes of pea seedlings ( 3 )  and of reticulocytes (4). 
The same is true of 
The low triphosphatase activity in the supernatant has been previously 
noted (1). 
due to small  amounts of incompletely sedimented particles, since when an 
It seems likely, moreover, that most of this activity is still 
aliquot of "supernatant" (previously centrifuged for 4 hours at 145,000 g 
in a Spinco rotor No. 50, and upper two thirds taken) was analyzed on a 
SDG, each of the triphosphatase activities sedimented in a series of w e l l -  
defined peaks (Fig. 6). While the positions of the peaks were the same 
for all activities, the activity of each enzyme relative to the others was 
greatly different from peak to peak. It should also be pointed out that 
since the bulk of the protein stayed essentially on top of the gradient 
(Fig. 2), the specific activity (per mg protein) of the sedimenting enzyme 
peaks was very high. 
RNA nor the protein profiles alone (Figs. 1 and 2) showed any signs of 
Another noteworthy fact is that, whereas neither the 
resolution, a number of components were discernible in the RNA to protein 
ratio profile of the supernatant (Fig. 3). The valleys (protein-rich 
components) in the latter correspond to the peaks of enzyme activity. 
Fig. 7 shows the distribution of each of the four triphosphatases 
in the reconstituted system compared with that of the individual compnents 
- 5 -  1 
on the one hand, and with that of (he crude extract ,  on the  other.  Each 
of the  four enzymes behaves different ly .  Most remarkable i s  the almost 
t o t a l  loss of the CTPase a c t i v i t y  of the  crude extract  upon purif icat ion 
of the ribosomal system. A large portion of the  t o t a l  ATPase a c t i v i t y  
is also los t ,  but some remains; note, however, that the supernatant seems 
t o  inh ib i t  the  ATPese ac t iv i ty  i n  the ribosomes, since the a c t i v i t y  of the 
reconsti tuted system i s  lower than t h a t  of the ribosomes. 
of the GliPase is  in te res t ing  i n  that, although it can be readi ly  washed 
off the ribosomes, it can a l so  be readsorbed from the supernatant. Finally, 
very little UTPase is los t ,  so t ha t  the enzymatic prof i le  of the reconsti tuted 
system i e  remarkably similar t o  t h a t  of the crude ex t rac t  i n  terms of t o t a l  
ac t iv i ty ,  although the  reconstituted system i s  more ac t ive  i n  terms of 
a c t i v i t y  per mg protein.  
The behaviour 
The loss  o r  enrichment of the enzymes r e l a t ive  t o  t h a t  of t o t a l  protein 
i s  brought out more c l ea r ly  i n  Fig. 8, which shows a comparison of spec i f ic  
a c t i v i t i e s .  Besides the r e l a t ive  enrichment i n  UTPase ac t iv i ty ,  it is  
par t icu lar ly  noteworthy tha t  t h e  GTPase a c t i v i t y  i n  the purif ied system i s  
almost exact ly  equivalent t o  t h a t  of the unfractionated ex t rac t  i n  terms 
of spec i f ic  ac t iv i ty .  
DISCUSSION 
The importance of the present resu l t s ,  besides strongly substant ia t ing 
the  claim of the previous paper (1) t h a t  the four triphosphatase a c t i v i t i e s  
res ide i n  physically d i s t i n c t  en t i t i es ,  l i es  i n  showing t h a t  the protein 
l o s t  from the ribosomes i n  the course of t h e i r  purif icat ion i s  qua l i ta t ive ly  
d i f f e ren t  from e i t h e r  t h e  supernatant o r  the  remaining ribosomal proteins.  
This finding i s  of relevance t o  studies on amino acid incorporation - i n  vitro,  
qu i te  regardless of any functional importance of these par t icu lar  
-* . 
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.. 
triphosphatases, because it shows up the possibility that other enzymes 
of potential importance m i g h t  be lost in the wash fluids and cannot be 
replaced by the supernatant. 
Moreover, the fact that GTPase lost on washing is readsorbed to its 
original level of specific activity when washed ribosomes are mixed with 
supernatant, whereas CTPase, although present in the supernatant in comparable 
amounts to CrPPase (cf. Fig. 6), is not readsorbed, shows that washing 
destroyed the binding site for one, but not the other enzyme. 
solubilization of the enzymes by ribonuclease (1) or pre-incubation of 
the crude extract (5) suggests that the binding of the enzymes is mediated 
by RINA, it further follows that the RNA, as w e l l  as the protein, lost in 
Since the 
the washes is qualitatively different from the remaining ribosomal RNA 
and, similarly, cannot be replenished from the supernatant. 
With these results, together with those in the two preceding papers 
(1, 2), the present study succeeded in its original objective of showing 
that many possible and previously unrecognized differences exist between 
ribosomal preparations which look identical by conventional sucrose density 
gradient analysis. The next step will be to determine the relevance, if 
any, of some of these changes to the mechanism of protein synthesis. 
ACKNOWLEXGEMEWTS 
are indebted t o  the Misses Sara P .  Goolsby and Es-elle Cook for 
help with the assays, and to Drs. Hardin B. Jones and Thomas H. Jukes 
for their  interest and financial assistance through a grant from the 
National Aeronautics and Space Agency (NSG479). 
The main support for t h i s  work was through NSF Grant G19532 and 
NIH G r a n t  GM-0792442. 
REFERENCES 
1. I. D. Raacke, Biochim. Biophys. Acta. 
2. 1. D. Raacke, Biochim. Biophys. Acta. 
3 .  I. D. Raacke, i n  preparation. 
4. J. F i s l a  and I. D. Raacke, i n  preparation. 
5. I. D. Rartcke and J. Fiala, Proc. N a t l .  Acad. Sci. U.S., 51 - - - - - -  
(1964) 323. 
. 
LEGENDS FOR FIGURES 
.. 
Fig. 1. Distribution of RNA. 
The broken lines represent purified ribosomes and supernatant, 
respectively; o.... o.... crude extract; --e, reconstituted system. 
Fig. 2- Distribution of protein. Legends as in Fig. 1. 
Fig. 3. RNA to protein ratio profiles. Legends as in Fig. 1. 
Fig .  4. Mstribution of UTPase activity in a crude ribosomal pellet. 
The ribosomes were prepared, recycled once, and analyzed in 0.005 M Tris, 
FlNA to protein ratio was 0.7. pH 7.4, 0.005 M Mg(C1)2. 
represents the distribution of RNA. 
The broken line 
Fig. 5. 
acetate, 0.005 M Tris, pH 7.1 (RNA/protein 1.41). 
Enzymatic activities of ribosomes washed three times in 0.01 M Mg 
The broken line represents RNA; x... x., ATPase; &-A , UTPase. The 
upper part represents specific activities, the lower one, total activities. 
Fig. 6 .  Distribution of triphosphatase activities in the supernatant fraction. 
x --- x -, ATPase; o .... o .., GTPase; A-4- UTPase; and 0 - 0, 
CTPase. The fastest peak corresponds to about 60s. 
Fig. 7. Comparison of total triphosphatase activities of crude extract 
(0- 
and the reconstituted system (0- 0-). 
), purified ribosomes (A--A-), supernatant fraction (x -- x -- ), 
Fig. 8. Comparison of specific triphosphatase activities of crude extract. 
Legends as in Fig. 7. 
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Figure 8 


Fate of specific ribonucleoside triphosphatases on purification and reconstitution of the ribosomal system of escherichia coli

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